Methods and compositions for weed control

ABSTRACT

Novel compositions for use to enhance weed control. Specifically, the present invention provides for methods and compositions that modulate protoporphyrinogen IX oxidase in weed species. The present invention also provides for combinations of compositions and methods that enhance weed control.

This application claims benefit under 35USC §119(e) of U.S. provisionalapplication Ser. No. 61/534,086 filed Sep. 13, 2011, herein incorporatedby reference in it's entirety. The sequence listing that is contained inthe file named “40_(—)21(58640)B seq listing.txt”, which is 862,548bytes (measured in operating system MS-Windows) and was created on 4Sep. 2012, is filed herewith and incorporated herein by reference.

Table 1 is provided herewith as a part of this U.S. patent applicationvia the USPTO's EFS system in file named “40_(—)21(58640)Btable 1.doxc ”which is 61,788 bytes in size (measured in MS-Windows®). Table 1 (file“40_(—)21(58640)Btable1.doxc” comprises 71 sequences and is hereinincorporated by reference in its entirety.

Table 2 is provided herewith as a part of this U.S. patent applicationvia the USPTO's EFS system in file named “40_(—)21(58640)Btable2.doxc ”which is 161,884 bytes in size (measured in MS-Windows®). Table 2 (file“40_(—)21(58640)Btable2.doxc” comprises 1268 sequences and is hereinincorporated by reference in its entirety.

Table 3 is provided herewith as a part of this U.S. patent applicationvia the USPTO's EFS system in file named “40_(—)21(58640)Btable3.txt ”which is 41,120 bytes in size (measured in MS-Windows®). Table 2 (file“40_(—)21(58640)Btable3.txt” comprises 832 sequences and is hereinincorporated by reference in its entirety.

FIELD

The methods and compositions generally relates to the field of weedmanagement. More specifically, relates to protoporphyrinogen IX oxidase(PPG oxidase) genes in plants and compositions containing polynucleotidemolecules for modulating and/or regulating their expression. Furtherprovided are methods and compositions useful for weed control.

BACKGROUND

Weeds are plants that compete with cultivated plants in an agronomicenvironment and cost farmers billions of dollars annually in crop lossesand the expense of efforts to keep weeds under control. Weeds also serveas hosts for crop diseases and insect pests. The losses caused by weedsin agricultural production environments include decreases in crop yield,reduced crop quality, increased irrigation costs, increased harvestingcosts, reduced land value, injury to livestock, and crop damage frominsects and diseases harbored by the weeds. The principal means by whichweeds cause these effects are: 1) competing with crop plants for water,nutrients, sunlight and other essentials for growth and development, 2)production of toxic or irritant chemicals that cause human or animalhealth problem, 3) production of immense quantities of seed orvegetative reproductive parts or both that contaminate agriculturalproducts and perpetuate the species in agricultural lands, and 4)production on agricultural and nonagricultural lands of vast amounts ofvegetation that must be disposed of. Herbicide tolerant weeds are aproblem with nearly all herbicides in use, there is a need toeffectively manage these weeds. There are over 365 weed biotypescurrently identified as being herbicide resistant to one or moreherbicides by the Herbicide Resistance Action Committee (HRAC), theNorth American Herbicide Resistance Action Committee (NAHRAC), and theWeed Science Society of America (WSSA).

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain methods, compositions orresults. They may be better understood by reference to one or more ofthese drawings in combination with the detailed description of specificembodiments presented herein. The invention can be more fully understoodfrom the following description of the figures:

FIG. 1. Treatment of Amaranthus palmeri with ssDNA triggerpolynucleotides and PPG oxidase inhibitor herbicide, flumioxazin.

FIG. 2. Treatment of Amaranthus palmeri with ssDNA triggerpolynucleotides and PPG oxidase inhibitor herbicide, fomesafen.

FIG. 3. Treatment of Amranthus palmeri with pooled oligos and ssDNAtrigger polynucleotides and PPG oxidase inhibitor herbicide, Reflex®(fomesafen).

SUMMARY

In one aspect, the invention provides a method of plant controlcomprising an external application to a plant of a compositioncomprising a polynucleotide and a transfer agent, wherein thepolynucleotide is essentially identical or essentially complementary toa PPG oxidase gene sequence or fragment thereof, or to the RNAtranscript of said PPG oxidase gene sequence or fragment thereof,wherein said PPG oxidase gene sequence is selected from the groupconsisting of SEQ ID NO:1-71 or a polynucleotide fragment thereof,whereby the plant growth or development or reproductive ability isreduced or the weedy plant is made more sensitive to a PPG oxidaseinhibitor herbicide relative to a plant not treated with saidcomposition. In this manner, plants that have become resistant to theapplication of PPG oxidase inhibitor contanining herbicides may be mademore susceptible to the herbicidal effects of a PPG oxidase inhibitorcontaining herbicide, thus potentiating the effect of the herbicide. Thepolynucleotide fragment is at least 18 contiguous nucleotides, at least19 contiguous nucleotides, at least 20 contiguous nucleotides or atleast 21 contiguous nucleotides in length and at least 85 percentidentical to a PPG oxidase gene sequence selected from the groupconsisting of SEQ ID NO:1-71 and the transfer agent is an organosiliconecomposition or compound. The polynucleotide fragment can also be senseor anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids. Thecomposition can include more than one polynucleotide fragments, and thecomposition can include a PPG oxidase inhibitor herbicide and/or otherherbicides that enhance the weed control activity of the composition.

In another aspect, polynucleotide molecules and methods for modulatingPPG oxidase gene expression in weedy plant species are provided. Themethod reduces, represses or otherwise delays expression of a PPGoxidase gene in a weedy plant comprising an external application to aweedy plant of a composition comprising a polynucleotide and a transferagent, wherein the polynucleotide is essentially identical oressentially complementary to a PPG oxidase gene sequence or fragmentthereof, or to the RNA transcript of the PPG oxidase gene sequence orfragment thereof, wherein the PPG oxidase gene sequence is selected fromthe group consisting of SEQ ID NO:1-71 or a polynucleotide fragmentthereof. The polynucleotide fragment fragment is at least 18 contiguousnucleotides, at least 19 contiguous nucleotides, at least 20 contiguousnucleotides at least 21 contiguous nucleotides in length and at least 85percent identical to a PPG oxidase gene sequence selected from the groupconsisting of SEQ ID NO:1-71 and the transfer agent is an organosiliconecompound. The polynucleotide fragment can also be sense or anti-sensessDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.

In a further aspect, the polynucleotide molecule containing compositionmay be combined with other herbicidal compounds to provide additionalcontrol of unwanted plants in a field of cultivated plants.

In a further aspect, the polynucleotide molecule composition may becombined with any one or more additional agricultural chemicals, suchas, insecticides, fungicides, nematocides, bactericides, acaricides,growth regulators, chemosterilants, semiochemicals, repellents,attractants, pheromones, feeding stimulants, biopesticides, microbialpesticides or other biologically active compounds to form amulti-component pesticide giving an even broader spectrum ofagricultural protection.

DETAILED DESCRIPTION

Provided are methods and compositions containing a polynucleotide thatprovide for regulation, repression or delay of PPG oxidase(protoporphyrinogen IX oxidase) gene expression and enhanced control ofweedy plant species amd importantly PPG oxidase inhibitor resistant weedbiotypes. Aspects of the method can be applied to manage various weedyplants in agronomic and other cultivated environments.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Where a term is provided in the singular, theinventors also contemplate aspects of the invention described by theplural of that term.

By “non-transcribable” polynucleotides is meant that the polynucleotidesdo not comprise a complete polymerase II transcription unit.

As used herein “solution” refers to homogeneous mixtures andnon-homogeneous mixtures such as suspensions, colloids, micelles, andemulsions.

Weedy plants are plants that compete with cultivated plants, those ofparticular importance include, but are not limited to important invasiveand noxious weeds and herbicide resistant biotypes in crop production,such as, Amaranthus species—A. albus, A. blitoides, A. hybridus, A.palmeri, A. powellii, A. retroflexus, A. spinosus, A. tuberculatus, andA. viridis; Ambrosia species—A. trifida, A. artemisifolia;Loliumspecies—L. multiflorum, L. rigidium, L perenne; Digitariaspecies—D. insularis; Euphorbia species—E. heterophylla; Kochiaspecies—K. scoparia; Sorghum species—S. halepense; Conyza species—C.bonariensis, C. canadensis, C. sumatrensis; Chloris species—C. truncate;Echinochola species—E. colona, E. crus-galli; Eleusine species—E.indica; Poa species—P. annua; Plantago species—P. lanceolata; Avenaspecies—A. fatua; Chenopodium species—C. album; Setaria species—S.viridis, Abutilon theophrasti, Ipomoea species, Sesbania, species,Cassia species, Sida species, Brachiaria, species and Solanum species.

Additional weedy plant species found in cultivated areas includeAlopecurus myosuroides, Avena sterilis, Avena sterilis ludoviciana,Brachiaria plantaginea, Bromus diandrus, Bromus rigidus, Cynosurusechinatus, Digitaria ciliaris, Digitaria ischaemum, Digitariasanguinalis, Echinochloa oryzicola, Echinochloa phyllopogon, Eriochloapunctata, Hordeum glaucum, Hordeum leporinum, Ischaemum rugosum,Leptochloa chinensis, Lolium persicum, Phalaris minor, Phalarisparadoxa, Rottboellia exalta, Setaria faberi, Setaria viridis var,robusta-alba schreiber, Setaria viridis var, robusta-purpurea, Snowdeniapolystachea, Sorghum sudanese, Alisma plantago-aquatica, Amaranthuslividus, Amaranthus quitensis, Ammania auriculata, Ammania coccinea,Anthemis cotula, Apera spica-venti, Bacopa rotundifolia, Bidens pilosa,Bidens subalternans, Brassica tournefortii, Bromus tectorum, Camelinamicrocarpa, Chrysanthemum coronarium, Cuscuta campestris, Cyperusdifformis, Damasonium minus, Descurainia sophia, Diplotaxis tenuifolia,Echium plantagineum, Elatine triandra var, pedicellate, Euphorbiaheterophylla, Fallopia convolvulus, Fimbristylis miliacea, Galeopsistetrahit, Galium spurium, Helianthus annuus, Iva xanthifolia, Ixophorusunisetus, Ipomoea indica, Ipomoea purpurea, Ipomoea sepiaria, Ipomoeaaquatic, Ipomoea triloba, Lactuca serriola, Limnocharis flava,Limnophila erecta, Limnophila sessiliflora, Lindernia dubia, Linderniadubia var, major, Lindernia micrantha, Lindernia procumbens,Mesembryanthemum crystallinum, Monochoria korsakowii, Monochoriavaginalis, Neslia paniculata, Papaver rhoeas, Parthenium hysterophorus,Pentzia suffruticosa, Phalaris minor, Raphanus raphanistrum, Raphanussativus, Rapistrum rugosum, Rotala indica var, uliginosa, Sagittariaguyanensis, Sagittaria montevidensis, Sagittaria pygmaea, Salsolaiberica, Scirpus juncoides var, ohwianus, Scirpus mucronatus, Setarialutescens, Sida spinosa, Sinapis arvensis, Sisymbrium orientale,Sisymbrium thellungii, Solanum ptycanthum, Sonchus aspen, Sonchusoleraceus, Sorghum bicolor, Stellaria media, Thlaspi arvense, Xanthiumstrumarium, Arctotheca calendula, Conyza sumatrensis, Crassocephalumcrepidiodes, Cuphea carthagenenis, Epilobium adenocaulon, Erigeronphiladelphicus, Landoltia punctata, Lepidium virginicum, Monochoriakorsakowii, Solanum americanum, Solanum nigrum, Vulpia bromoides,Youngia japonica, Hydrilla verticillate, Carduus nutans, Carduuspycnocephalus, Centaurea solstitialis, Cirsium arvense, Commelinadiffusa, Convolvulus arvensis, Daucus carota, Digitaria ischaemum,Echinochloa crus-pavonis, Fimbristylis miliacea, Galeopsis tetrahit,Galium spurium, Limnophila erecta, Matricaria perforate, Papaver rhoeas,Ranunculus acris, Soliva sessilis, Sphenoclea zeylanica, Stellariamedia, Nassella trichotoma, Stipa neesiana, Agrostis stolonifera,Polygonum aviculare, Alopecurus japonicus, Beckmannia syzigachne, Bromustectorum, Chloris inflate, Echinochloa erecta, Portulaca oleracea, andSenecio vulgaris. It is believed that all plants contain anprotoporphyrinogen IX oxidase gene in their genome, the sequence ofwhich can be isolated and polynucleotides made according to the methodsof the present invention that are useful for regulating, suppressing ordelaying the expression of the target PPG oxidase gene in the plants andthe growth or development of the treated plants.

Some cultivated plants may also be weedy plants when they occur inunwanted environments. Transgenic crops with one or more herbicidetolerances will need specialized methods of management to control weedsand volunteer crop plants and to target the herbicide tolerancetransgene as necessary to permit the treated plants to become sensitiveto the herbicide.

A “trigger” or “trigger polynucleotide” is a polynucleotide moleculethat is homologous or complementary to a target gene polynucleotide. Thetrigger polynucleotide molecules modulate expression of the target genewhen topically applied to a plant surface with a transfer agent, wherebya plant treated with said composition has its growth or development orreproductive ability regulated, suppressed or delayed or said plant ismore sensitive to a PPG oxidase inhibitor herbicide or mitosis inhibitorherbicide as a result of said polynucleotide containing compositionrelative to a plant not treated with a composition containing thetrigger molecule. Trigger polynucleotides disclosed herein are generallydescribed in relation to the target gene sequence and maybe used in thesense (homologous) or antisense (complementary) orientation as singlestranded molecules or comprise both strands as double stranded moleculesor nucleotide variants and modified nucleotides thereof depending on thevarious regions of a gene being targeted.

It is contemplated that the composition of the present invention willcontain multiple polynucleotides and herbicides that include but notlimited to PPG oxidase gene trigger polynucleotides and a PPG oxidaseinhibitor herbicide and anyone or more additional herbicide target genetrigger polynucleotides and the related herbicides and anyone or moreadditional essential gene trigger polynucleotides. Essential genes aregenes in a plant that provide key enzymes or other proteins, forexample, a biosynthetic enzyme, metabolizing enzyme, receptor, signaltransduction protein, structural gene product, transcription factor, ortransport protein; or regulating RNAs, such as, microRNAs, that areessential to the growth or survival of the organism or cell or involvedin the normal growth and development of the plant (Meinke, et al.,Trends Plant Sci. 2008 Sep;13(9):483-91). The suppression of anessential gene enhances the effect of a herbicide that affects thefunction of a gene product different than the suppressed essential gene.The compositions of the present invention can include various triggerpolynucleotides that modulate the expression of an essential gene otherthan PPG oxidase.

Herbicides for which transgenes for plant tolerance have beendemonstrated and the method of the present invention can be applied,include but are not limited to: auxin-like herbicides, glyphosate,glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon,dicamba, cyclohezanedione, protoporphyrionogen oxidase inhibitors,4-hydroxyphenyl-pyruvate-dioxygenase inhibitors herbicides. For example,transgenes and their polynucleotide molecules that encode proteinsinvolved in herbicide tolerance are known in the art, and include, butare not limited to an 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), for example, as more fully described in U.S. Pat. Nos.7,807,791 (SEQ ID NO:5); 6,248,876 B1; 5,627,061; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,462,910; 5,188,642; 4,940,835; 5,866,775;6,225,114 B1; 6,130,366; 5,460,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; U.S. Pat. No. Re. 36,449; U.S. Pat. Nos. RE 37,287 E; and5,491,288; tolerance to sulfonylurea and/or imidazolinone, for example,as described more fully in U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,746,180; 5,304,732; 4,761,373; 5,346,107;5,928,937; and 5,378,824; and international publication WO 96/33270;tolerance to hydroxyphenylpyruvatedioxygenases inhibitiong herbicides inplants are described in U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and6,069,115; US Pat.Pub. 20110191897 and U.S. Pat. No. 7,462,379 SEQ IDNO:3; U.S. Pat. No. 7,935,869; U.S. Pat. No. 7,304,209, SEQ ID NO:1, 3,5and 15; aryloxyalkanoate dioxygenase polynucleotides, which confertolerance to 2,4-D and other phenoxy auxin herbicides as well as toaryloxyphenoxypropionate herbicides as described, for example, inWO2005/107437; U.S. Pat. No. 7,838,733 SEQ ID NO:5;) anddicamba-tolerance polynucleotides as described, for example, in Hermanet al. (2005) J. Biol. Chem. 280: 24759-24767. Other examples ofherbicide-tolerance traits include those conferred by polynucleotidesencoding an exogenous phosphinothricin acetyltransferase, as describedin U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,468; 5,874,265; 5,919,675;5,561,236; 5,648,477; 5,646,024; 6,177,616; and 5,879,903. Plantscontaining an exogenous phosphinothricin acetyltransferase can exhibitimproved tolerance to glufosinate herbicides, which inhibit the enzymeglutamine synthase. Additionally, herbicide-tolerance polynucleotidesinclude those conferred by polynucleotides conferring alteredprotoporphyrinogen oxidase (protox) activity, as described in U.S. Pat.Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and WO 01/12825. Plantscontaining such polynucleotides can exhibit improved tolerance to any ofa variety of herbicides which target the protox enzyme (also referred toas protox inhibitors). Polynucleotides encoding a glyphosateoxidoreductase and a glyphosate-N-acetyl transferase (GOX described inU.S. Pat. No. 5,463,175 and GAT described in U.S. Patent publication20030083480, dicamba monooxygenase U.S. Patent publication 20030135879,all of which are incorporated herein by reference); a polynucleotidemolecule encoding bromoxynil nitrilase (Bxn described in U.S. Pat. No.4,810,648 for Bromoxynil tolerance, which is incorporated herein byreference); a polynucleotide molecule encoding phytoene desaturase(crtI) described in Misawa et al, (1993) Plant J. 4:833-840 and Misawaet al, (1994) Plant J. 6:481-489 for norflurazon tolerance; apolynucleotide molecule encoding acetohydroxyacid synthase (AHAS, akaALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:468-2193for tolerance to sulfonylurea herbicides; and the bar gene described inDeBlock, et al. (1987) EMBO J. 6:2513-2519 for glufosinate and bialaphostolerance. The transgenic coding regions and regulatory elements of theherbicide tolerance genes are targets in which polynucleotide triggersand herbicides can be included in the composition of the presentinvention.

The composition includes a component that is a PPG oxidase inhibitorherbicide, which include but is not limited to acifluorfen-Na, bifenox,chlomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen,oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin,flumiclorac-pentyl, fluthiacet-methyl, thidiazimin, oxadiazon,oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone.Benzfendizone, butafenacil, pyrazogyl, and profluazol.

Numerous additional herbicides with similar or different modes of action(herein referred to as co-herbicides) are available that can be added tothe composition, for example, members of the herbicide families thatinclude but are not limited to amide herbicides, aromatic acidherbicides, arsenical herbicides, benzothiazole herbicides,benzoylcyclohexanedione herbicides, benzofuranyl alkylsulfonateherbicides, carbamate herbicides, cyclohexene oxime herbicides,cyclopropylisoxazole herbicides, dicarboximide herbicides,dinitroaniline herbicides, dinitrophenol herbicides, diphenyl etherherbicides, dithiocarbamate herbicides, halogenated aliphaticherbicides, imidazolinone herbicides, inorganic herbicides, nitrileherbicides, organophosphorus herbicides, oxadiazolone herbicides,oxazole herbicides, phenoxy herbicides, phenylenediamine herbicides,pyrazole herbicides, pyridazine herbicides, pyridazinone herbicides,pyridine herbicides, pyrimidinediamine herbicides,pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides,thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides,triazine herbicides, triazinone herbicides, triazole herbicides,triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides,and urea herbicides. In particular, the rates of use of the addedherbicides can be reduced in compositions comprising thepolynucleotides. Use rate reductions of the additional added herbicidescan be 10-25 percent, 26-50 percent, 51-75 percent or more can beachieved that enhance the activity of the polynucleotides and herbicidecomposition and is contemplated. Representative co-herbicides of thefamilies include but are not limited to acetochlor, acifluorfen,acifluorfen-sodium, aclonifen, acrolein, alachlor, alloxydim, allylalcohol, ametryn, amicarbazone, amidosulfuron, aminopyralid, amitrole,ammonium sulfamate, anilofos, asulam, atraton, atrazine, azimsulfuron,BCPC, beflubutamid, benazolin, benfluralin, benfuresate, bensulfuron,bensulfuron-methyl, bensulide, bentazone, benzfendizone, benzobicyclon,benzofenap, bifenox, bilanafos, bispyribac, bispyribac-sodium, borax,bromacil, bromobutide, bromoxynil, butachlor, butafenacil, butamifos,butralin, butroxydim, butylate, cacodylic acid, calcium chlorate,cafenstrole, carbetamide, carfentrazone, carfentrazone-ethyl, CDEA,CEPC, chlorflurenol, chlorflurenol-methyl, chloridazon, chlorimuron,chlorimuron-ethyl, chloroacetic acid, chlorotoluron, chlorpropham,chlorsulfuron, chlorthal, chlorthal-dimethyl, cinidon-ethyl,cinmethylin, cinosulfuron, cisanilide, clethodim, clodinafop,clodinafop-propargyl, clomazone, clomeprop, clopyralid, cloransulam,cloransulam-methyl, CMA, 4-CPB, CPMF, 4-CPP, CPPC, cresol, cumyluron,cyanamide, cyanazine, cycloate, cyclosulfamuron, cycloxydim, cyhalofop,cyhalofop-butyl, 2,4-D, 3,4-DA, daimuron, dalapon, dazomet, 2,4-DB,3,4-DB, 2,4-DEB, desmedipham, dicamba, dichlobenil,ortho-dichlorobenzene, para-dichlorobenzene, dichlorprop, dichlorprop-P,diclofop, diclofop-methyl, diclosulam, difenzoquat, difenzoquatmetilsulfate, diflufenican, diflufenzopyr, dimefuron, dimepiperate,dimethachlor, dimethametryn, dimethenamid, dimethenamid-P, dimethipin,dimethylarsinic acid, dinitramine, dinoterb, diphenamid, diquat, diquatdibromide, dithiopyr, diuron, DNOC, 3,4-DP, DSMA, EBEP, endothal, EPTC,esprocarb, ethalfluralin, ethametsulfuron, ethametsulfuron-methyl,ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-P,fenoxaprop-P-ethyl, fentrazamide, ferrous sulfate, flamprop-M,flazasulfuron, florasulam, fluazifop, fluazifop-butyl, fluazifop-P,fluazifop-P-butyl, flucarbazone, flucarbazone-sodium, flucetosulfuron,fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam,flumiclorac, flumiclorac-pentyl, flumioxazin, fluometuron,fluoroglycofen, fluoroglycofen-ethyl, flupropanate, flupyrsulfuron,flupyrsulfuron-methyl-sodium, flurenol, fluridone, fluorochloridone,fluoroxypyr, flurtamone, fluthiacet, fluthiacet-methyl, fomesafen,foramsulfuron, fosamine, glufosinate, glufosinate-ammonium, glyphosate,halosulfuron, halosulfuron-methyl, haloxyfop, haloxyfop-P, HC-252,hexazinone, imazamethabenz, imazamethabenz-methyl, imazamox, imazapic,imazapyr, imazaquin, imazethapyr, imazosulfuron, indanofan, iodomethane,iodosulfuron, iodosulfuron-methyl-sodium, ioxynil, isoproturon, isouron,isoxaben, isoxachlortole, isoxaflutole, karbutilate, lactofen, lenacil,linuron, MAA, MAMA, MCPA, MCPA-thioethyl, MCPB, mecoprop, mecoprop-P,mefenacet, mefluidide, mesosulfuron, mesosulfuron-methyl, mesotrione,metam, metamifop, metamitron, metazachlor, methabenzthiazuron,methylarsonic acid, methyldymron, methyl isothiocyanate, metobenzuron,metolachlor, S-metolachlor, metosulam, metoxuron, metribuzin,metsulfuron, metsulfuron-methyl, MK-66, molinate, monolinuron, MSMA,naproanilide, napropamide, naptalam, neburon, nicosulfuron, nonanoicacid, norflurazon, oleic acid (fatty acids), orbencarb, orthosulfamuron,oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone,oxyfluorfen, paraquat, paraquat dichloride, pebulate, pendimethalin,penoxsulam, pentachlorophenol, pentanochlor, pentoxazone, pethoxamid,petrolium oils, phenmedipham, phenmedipham-ethyl, picloram, picolinafen,pinoxaden, piperophos, potassium arsenite, potassium azide,pretilachlor, primisulfuron, primisulfuron-methyl, prodiamine,profluazol, profoxydim, prometon, prometryn, propachlor, propanil,propaquizafop, propazine, propham, propisochlor, propoxycarbazone,propoxycarbazone-sodium, propyzamide, prosulfocarb, prosulfuron,pyraclonil, pyraflufen, pyraflufen-ethyl, pyrazolynate, pyrazosulfuron,pyrazosulfuron-ethyl, pyrazoxyfen, pyribenzoxim, pyributicarb,pyridafol, pyridate, pyriftalid, pyriminobac, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, quinclorac, quinmerac,quinoclamine, quizalofop, quizalofop-P, rimsulfuron, sethoxydim,siduron, simazine, simetryn, SMA, sodium arsenite, sodium azide, sodiumchlorate, sulcotrione, sulfentrazone, sulfometuron, sulfometuron-methyl,sulfosate, sulfosulfuron, sulfuric acid, tar oils, 2,3,6-TBA, TCA,TCA-sodium, tebuthiuron, tepraloxydim, terbacil, terbumeton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thifensulfuron,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron,tribenuron-methyl, tricamba, triclopyr, trietazine, trifloxysulfuron,trifloxysulfuron-sodium, trifluralin, triflusulfuron,triflusulfuron-methyl, trihydroxytriazine, tritosulfuron,[3-[2-chloro-4-fluoro-54-methyl-6-trifluoromethyl-2,4-dioxo-, 2,3,44-etrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetic acid ethyl ester(CAS RN 353292-3-6), 4-[(4,5-dihydro-3-methoxy-4-methyl-5-oxo)-H-,2,4-triazol-ylcarbonyl-sulfamoyl]-5-methylthiophene-3-carboxylic acid(BAY636), BAY747 (CAS RN 33504-84-2), topramezone (CAS RN 2063-68-8),4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoro-methyl)-3-pyridi-nyl]carbonyl]-bicyclo[3.2.]oct-3-en-2-one(CAS RN 35200-68-5), and4-hydroxy-3-[[2-(3-methoxypropyl)-6-(difluoromethyl)-3-pyridinyl]carbon-y1]-bicyclo[3.2.]oct-3-en-2-one. Additionally, including herbicidalcompounds of unspecified modes of action as described in CN101279950A,CN101279951A, DE10000600A1, DE10116399A1, DE102004054666A1,DE102005014638A1, DE102005014906A1, DE102007012168A1, DE102010042866A1,DE10204951A1, DE10234875A1, DE10234876A1, DE10256353A1, DE10256354A1,DE10256367A1, EP1157991A2, EP1238586A1, EP2147919A1, EP2160098A2,JP03968012B2, JP2001253874A, JP2002080454A, JP2002138075A,JP2002145707A, JP2002220389A, JP2003064059A, JP2003096059A,JP2004051628A, JP2004107228A, JP2005008583A, JP2005239675A,JP2005314407A, JP2006232824A, JP2006282552A, JP2007153847A,JP2007161701A, JP2007182404A, JP2008074840A, JP2008074841A,JP2008133207A, JP2008133218A, JP2008169121A, JP2009067739A,JP2009114128A, JP2009126792A, JP2009137851A, US20060111241A1,US20090036311A1, US20090054240A1, US20090215628A1, US20100099561A1,US20100152443A1, US20110105329A1, US20110201501A1, WO2001055066A2,WO2001056975A1, WO2001056979A1, WO2001090071A2, WO2001090080A1,WO2002002540A1 , WO2002028182A1, WO2002040473A1, WO2002044173A2,WO2003000679A2, WO2003006422A1 , WO2003013247A1, WO2003016308A1,WO2003020704A1, WO2003022051A1, WO2003022831A1, WO2003022843A1,WO2003029243A2, WO2003037085A1, WO2003037878A1, WO2003045878A2,WO2003050087A2, WO2003051823A1, WO2003051824A1, WO2003051846A2,WO2003076409A1, WO2003087067A1, WO2003090539A1, WO2003091217A1,WO2003093269A2, WO2003104206A2, WO2004002947A1, WO2004002981A2,WO2004011429A1, WO2004029060A1, WO2004035545A2, WO2004035563A1,WO2004035564A1, WO2004037787A1, WO2004067518A1, WO2004067527A1,WO2004077950A1, WO2005000824A1, WO2005007627A1, WO2005040152A1,WO2005047233A1, WO2005047281A1, WO2005061443A2, WO2005061464A1,WO2005068434A1, WO2005070889A1, WO2005089551A1, WO2005095335A1,WO2006006569A1, WO2006024820A1, WO2006029828A1, WO2006029829A1,WO2006037945A1, WO2006050803A1, WO2006090792A1, WO2006123088A2,WO2006125687A1, WO2006125688A1, WO2007003294A1, WO2007026834A1,WO2007071900A1, WO2007077201A1, WO2007077247A1, WO2007096576A1,WO2007119434A1, WO2007134984A1, WO2008009908A1, WO2008029084A1,WO2008059948A1, WO2008071918A1, WO2008074991A1, WO2008084073A1,WO2008100426A2, WO2008102908A1, WO2008152072A2, WO2008152073A2,WO2009000757A1, WO2009005297A2, WO2009035150A2, WO2009063180A1,WO2009068170A2, WO2009068171A2, WO2009086041A1, WO2009090401A2,WO2009090402A2, WO2009115788A1, WO2009116558A1, WO2009152995A1,WO2009158258A1, WO2010012649A1, WO2010012649A1, WO2010026989A1,WO2010034153A1, WO2010049270A1, WO2010049369A1, WO2010049405A1,WO2010049414A1, WO2010063422A1, WO2010069802A1, WO2010078906A2,WO2010078912A1, WO2010104217A1, WO2010108611A1, WO2010112826A3,WO2010116122A3, WO2010119906A1, WO2010130970A1, WO2011003776A2,WO2011035874A1, WO2011065451A1, all of which are incorporated herein byreference.

An agronomic field in need of plant control is treated by application ofthe composition directly to the surface of the growing plants, such asby a spray. For example, the method is applied to control weeds in afield of crop plants by spraying the field with the composition.. Thecomposition can be provided as a tank mix, a sequential treatment ofcomponents (generally the polynucleotide containing composition followedby the herbicide), or a simultaneous treatment or mixing of one or moreof the components of the composition from separate containers. Treatmentof the field can occur as often as needed to provide weed control andthe components of the composition can be adjusted to target specificweed species or weed families through utilization of specificpolynucleotides or polynucleotide compositions capable of selectivelytargeting the specific species or plant family to be controlled. Thecomposition can be applied at effective use rates according to the timeof application to the field, for example, preplant, at planting, postplanting, post harvest. PPG oxidase inhibitor herbicides can be appliedto a field at rates of 100 to 500 g ai/ha (active ingredient perhectare) or more. The polynucleotides of the composition can be appliedat rates of 1 to 30 grams per acre depending on the number of triggermolecules needed for the scope of weeds in the field.

Crop plants in which weed control is needed include but are not limitedto, i) corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane,rice, and wheat; ii) vegetable plants including, but not limited to,tomato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant,cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweetcorn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish,Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii)culinary plants including, but not limited to, basil, parsley, coffee,or tea; or , iv) fruit plants including but not limited to apple, pear,cherry, peach, plum, apricot, banana, plantain, table grape, wine grape,citrus, avocado, mango, or berry; v) a tree grown for ornamental orcommercial use, including, but not limited to, a fruit or nut tree; or,vi) an ornamental plant (e. g., an ornamental flowering plant or shrubor turf grass). The methods and compositions provided herein can also beapplied to plants produced by a cutting, cloning, or grafting process(i. e., a plant not grown from a seed) include fruit trees and plantsthat include, but are not limited to, citrus, apples, avocados,tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well asvarious ornamental plants.

Pesticidal Mixtures

The polynucleotide compositions may also be used as mixtures withvarious agricultural chemicals and/or insecticides, miticides andfungicides, pesticidal and biopesticidal agents. Examples include butare not limited to azinphos-methyl, acephate, isoxathion, isofenphos,ethion, etrimfos, oxydemeton-methyl, oxydeprofos, quinalphos,chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, cyanophos,dioxabenzofos, dichlorvos, disulfoton, dimethylvinphos, dimethoate,sulprofos, diazinon, thiometon, tetrachlorvinphos, temephos,tebupirimfos, terbufos, naled, vamidothion, pyraclofos, pyridafenthion,pirimiphos-methyl, fenitrothion, fenthion, phenthoate, flupyrazophos,prothiofos, propaphos, profenofos, phoxime, phosalone, phosmet,formothion, phorate, malathion, mecarbam, mesulfenfos, methamidophos,methidathion, parathion, methyl parathion, monocrotophos, trichlorphon,EPN, isazophos, isamidofos, cadusafos, diamidaphos, dichlofenthion,thionazin, fenamiphos, fosthiazate, fosthietan, phosphocarb, DSP,ethoprophos, alanycarb, aldicarb, isoprocarb, ethiofencarb, carbaryl,carbosulfan, xylylcarb, thiodicarb, pirimicarb, fenobucarb,furathiocarb, propoxur, bendiocarb, benfuracarb, methomyl, metolcarb,XMC, carbofuran, aldoxycarb, oxamyl, acrinathrin, allethrin,esfenvalerate, empenthrin, cycloprothrin, cyhalothrin,gamma-cyhalothrin, lambda-cyhalothrin, cyfluthrin, beta-cyfluthrin,cypermethrin, alpha-cypermethrin, zeta-cypermethrin, silafluofen,tetramethrin, tefluthrin, deltamethrin, tralomethrin, bifenthrin,phenothrin, fenvalerate, fenpropathrin, furamethrin, prallethrin,flucythrinate, fluvalinate, flubrocythrinate, permethrin, resmethrin,ethofenprox, cartap, thiocyclam, bensultap, acetamiprid, imidacloprid,clothianidin, dinotefuran, thiacloprid, thiamethoxam, nitenpyram,chlorfluazuron, diflubenzuron, teflubenzuron, triflumuron, novaluron,noviflumuron, bistrifluoron, fluazuron, flucycloxuron, flufenoxuron,hexaflumuron, lufenuron, chromafenozide, tebufenozide, halofenozide,methoxyfenozide, diofenolan, cyromazine, pyriproxyfen, buprofezin,methoprene, hydroprene, kinoprene, triazamate, endosulfan, chlorfenson,chlorobenzilate, dicofol, bromopropylate, acetoprole, fipronil,ethiprole, pyrethrin, rotenone, nicotine sulphate, BT (BacillusThuringiensis) agent, spinosad, abamectin, acequinocyl, amidoflumet,amitraz, etoxazole, chinomethionat, clofentezine, fenbutatin oxide,dienochlor, cyhexatin, spirodiclofen, spiromesifen, tetradifon,tebufenpyrad, binapacryl, bifenazate, pyridaben, pyrimidifen,fenazaquin, fenothiocarb, fenpyroximate, fluacrypyrim, fluazinam,flufenzin, hexythiazox, propargite, benzomate, polynactin complex,milbemectin, lufenuron, mecarbam, methiocarb, mevinphos, halfenprox,azadirachtin, diafenthiuron, indoxacarb, emamectin benzoate, potassiumoleate, sodium oleate, chlorfenapyr, tolfenpyrad, pymetrozine,fenoxycarb, hydramethylnon, hydroxy propyl starch, pyridalyl,flufenerim, flubendiamide, flonicamid, metaflumizole, lepimectin, TPIC,albendazole, oxibendazole, oxfendazole, trichlamide, fensulfothion,fenbendazole, levamisole hydrochloride, morantel tartrate, dazomet,metam-sodium, triadimefon, hexaconazole, propiconazole, ipconazole,prochloraz, triflumizole, tebuconazole, epoxiconazole, difenoconazole,flusilazole, triadimenol, cyproconazole, metconazole, fluquinconazole,bitertanol, tetraconazole, triticonazole, flutriafol, penconazole,diniconazole, fenbuconazole, bromuconazole, imibenconazole,simeconazole, myclobutanil, hymexazole, imazalil, furametpyr,thifluzamide, etridiazole, oxpoconazole, oxpoconazole fumarate,pefurazoate, prothioconazole, pyrifenox, fenarimol, nuarimol,bupirimate, mepanipyrim, cyprodinil, pyrimethanil, metalaxyl, mefenoxam,oxadixyl, benalaxyl, thiophanate, thiophanate-methyl, benomyl,carbendazim, fuberidazole, thiabendazole, manzeb, propineb, zineb,metiram, maneb, ziram, thiuram, chlorothalonil, ethaboxam, oxycarboxin,carboxin, flutolanil, silthiofam, mepronil, dimethomorph, fenpropidin,fenpropimorph, spiroxamine, tridemorph, dodemorph, flumorph,azoxystrobin, kresoxim-methyl, metominostrobin, orysastrobin,fluoxastrobin, trifloxystrobin, dimoxystrobin, pyraclostrobin,picoxystrobin, iprodione, procymidone, vinclozolin, chlozolinate,flusulfamide, dazomet, methyl isothiocyanate, chloropicrin,methasulfocarb, hydroxyisoxazole, potassium hydroxyisoxazole,echlomezol, D-D, carbam, basic copper chloride, basic copper sulfate,copper nonylphenolsulfonate, oxine copper, DBEDC, anhydrous coppersulfate, copper sulfate pentahydrate, cupric hydroxide, inorganicsulfur, wettable sulfur, lime sulfur, zinc sulfate, fentin, sodiumhydrogen carbonate, potassium hydrogen carbonate, sodium hypochlorite,silver, edifenphos, tolclofos-methyl, fosetyl, iprobenfos, dinocap,pyrazophos, carpropamid, fthalide, tricyclazole, pyroquilon, diclocymet,fenoxanil, kasugamycin, validamycin, polyoxins, blasticiden S,oxytetracycline, mildiomycin, streptomycin, rape seed oil, machine oil,benthiavalicarbisopropyl, iprovalicarb, propamocarb, diethofencarb,fluoroimide, fludioxanil, fenpiclonil, quinoxyfen, oxolinic acid,chlorothalonil, captan, folpet, probenazole, acibenzolar-S-methyl,tiadinil, cyflufenamid, fenhexamid, diflumetorim, metrafenone,picobenzamide, proquinazid, famoxadone, cyazofamid, fenamidone,zoxamide, boscalid, cymoxanil, dithianon, fluazinam, dichlofluanide,triforine, isoprothiolane, ferimzone, diclomezine, tecloftalam,pencycuron, chinomethionat, iminoctadine acetate, iminoctadinealbesilate, ambam, polycarbamate, thiadiazine, chloroneb, nickeldimethyldithiocarbamate, guazatine, dodecylguanidine-acetate,quintozene, tolylfluanid, anilazine, nitrothalisopropyl, fenitropan,dimethirimol, benthiazole, harpin protein, flumetover, mandipropamideand penthiopyrad.

Polynucleotides

As used herein, the term “DNA”, “DNA molecule”, “DNA polynucleotidemolecule” refers to a single-stranded DNA (ssDNA) or double-stranded DNA(dsDNA) molecule of genomic or synthetic origin, such as, a polymer ofdeoxyribonucleotide bases or a DNA polynucleotide molecule. As usedherein, the term “DNA sequence”, “DNA nucleotide sequence” or “DNApolynucleotide sequence” refers to the nucleotide sequence of a DNAmolecule. As used herein, the term “RNA”, “RNA molecule”, “RNApolynucleotide molecule” refers to a single-stranded RNA (ssRNA) ordouble-stranded RNA (dsRNA) molecule of genomic or synthetic origin,such as, a polymer of ribonucleotide bases that comprise single ordouble stranded regions. Unless otherwise stated, nucleotide sequencesin the text of this specification are given, when read from left toright, in the 5′ to 3′ direction. The nomenclature used herein is thatrequired by Title 37 of the United States Code of Federal Regulations§1.822 and set forth in the tables in WIPO Standard ST.25 (1998),Appendix 2, Tables 1 and 3.

As used herein, “polynucleotide” refers to a DNA or RNA moleculecontaining multiple nucleotides and generally refers both to“oligonucleotides” (a polynucleotide molecule of typically 50 or fewernucleotides in length) and polynucleotides of 51 or more nucleotides.Embodiments include compositions including oligonucleotides having alength of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers,22-mers, 23-mers, 24-mers, or 25-mers), for example, oligonucleotides ofTable 3 (SEQ ID NO:1382-2221) or fragments thereof or medium-lengthpolynucleotides having a length of 26 or more nucleotides(polynucleotides of 26, 27, 28, 29, 30, 46, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 260, about 270,about 280, about 290, or about 300 nucleotides), for example,oligonucleotides of Table 2 (SEQ ID NO:72-1381) or fragments thereof orlong polynucleotides having a length greater than about 300 nucleotides(for example, polynucleotides of between about 300 to about 400nucleotides, between about 400 to about 500 nucleotides, between about500 to about 600 nucleotides, between about 600 to about 700nucleotides, between about 700 to about 800 nucleotides, between about800 to about 900 nucleotides, between about 900 to about 1000nucleotides, between about 300 to about 500 nucleotides, between about300 to about 600 nucleotides, between about 300 to about 700nucleotides, between about 300 to about 800 nucleotides, between about300 to about 900 nucleotides, or about 1000 nucleotides in length, oreven greater than about 1000 nucleotides in length, for example up tothe entire length of a target gene including coding or non-coding orboth coding and non-coding portions of the target gene), for example,polynucleotides of Table 1 (SEQ ID NO:1-71), wherein the selectedpolynucleotides or fragments thereof homologous or complementary to SEQID NO:1-71 suppresses, represses or otherwise delay the expression ofthe target PPG oxidase gene. A target gene comprises any polynucleotidemolecule in a plant cell or fragment thereof for which the modulation ofthe expression of the target gene is provided by the methods andcompositions. Where a polynucleotide is double-stranded, its length canbe similarly described in terms of base pairs. Oligonucleotides andpolynucleotides can be made that are essentially identical oressentially complementary to adjacent genetic elements of a gene, forexample, spanning the junction region of an intron and exon, thejunction region of a promoter and a transcribed region, the junctionregion of a 5′ leader and a coding sequence, the junction of a 3′untranslated region and a coding sequence.

Polynucleotide compositions used in the various embodiments includecompositions including oligonucleotides or polynucleotides or a mixtureof both, including RNA or DNA or RNA/DNA hybrids or chemically modifiedoligonucleotides or polynucleotides or a mixture thereof. In someembodiments, the polynucleotide may be a combination of ribonucleotidesand deoxyribonucleotides, for example, synthetic polynucleotidesconsisting mainly of ribonucleotides but with one or more terminaldeoxyribonucleotides or synthetic polynucleotides consisting mainly ofdeoxyribonucleotides but with one or more terminaldideoxyribonucleotides. In some embodiments, the polynucleotide includesnon-canonical nucleotides such as inosine, thiouridine, orpseudouridine. In some embodiments, the polynucleotide includeschemically modified nucleotides. Examples of chemically modifiedoligonucleotides or polynucleotides are well known in the art; see, forexample, US Patent Publication 20110171287, US Patent Publication20110171176, and US Patent Publication 20110152353, US PatentPublication, 20110152346, US Patent Publication 20110160082, hereinincorporated by reference. For example, including but not limited to thenaturally occurring phosphodiester backbone of an oligonucleotide orpolynucleotide can be partially or completely modified withphosphorothioate, phosphorodithioate, or methylphosphonateinternucleotide linkage modifications, modified nucleoside bases ormodified sugars can be used in oligonucleotide or polynucleotidesynthesis, and oligonucleotides or polynucleotides can be labeled with afluorescent moiety (for example, fluorescein or rhodamine) or otherlabel (for example, biotin).

The polynucleotides can be single- or double-stranded RNA or single- ordouble-stranded DNA or double-stranded DNA/RNA hybrids or modifiedanalogues thereof, and can be of oligonucleotide lengths or longer. Inmore specific embodiments the polynucleotides that providesingle-stranded RNA in the plant cell are selected from the groupconsisting of (a) a single-stranded RNA molecule (ssRNA), (b) asingle-stranded RNA molecule that self-hybridizes to form adouble-stranded RNA molecule, (c) a double-stranded RNA molecule(dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) asingle-stranded DNA molecule that self-hybridizes to form adouble-stranded DNA molecule, and (f) a single-stranded DNA moleculeincluding a modified Pol III gene that is transcribed to an RNAmolecule, (g) a double-stranded DNA molecule (dsDNA), (h) adouble-stranded DNA molecule including a modified Pol III gene that istranscribed to an RNA molecule, (i) a double-stranded, hybridizedRNA/DNA molecule, or combinations thereof. In some embodiments thesepolynucleotides include chemically modified nucleotides or non-canonicalnucleotides. In some embodiments, the oligonucleotides may beblunt-ended or may comprise a 3′ overhang of from 1-5 nucleotides of atleast one or both of the strands. Other configurations of theoligonucleotide are known in the field and are contemplated herein. Inembodiments of the method the polynucleotides include double-strandedDNA formed by intramolecular hybridization, double-stranded DNA formedby intermolecular hybridization, double-stranded RNA formed byintramolecular hybridization, or double-stranded RNA formed byintermolecular hybridization. In one embodiment the polynucleotidesinclude single-stranded DNA or single-stranded RNA that self-hybridizesto form a hairpin structure having an at least partially double-strandedstructure including at least one segment that will hybridize to RNAtranscribed from the gene targeted for suppression. Not intending to bebound by any mechanism, it is believed that such polynucleotides are orwill produce single-stranded RNA with at least one segment that willhybridize to RNA transcribed from the gene targeted for suppression. Incertain other embodiments the polynucleotides further includes apromoter, generally a promoter functional in a plant, for example, a polII promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

The term “gene” refers to components that comprise chromosomal DNA,plasmid DNA, cDNA, intron and exon DNA, artificial DNA polynucleotide,or other DNA that encodes a peptide, polypeptide, protein, or RNAtranscript molecule, and the genetic elements flanking the codingsequence that are involved in the regulation of expression, such as,promoter regions, 5′ leader regions, 3′ untranslated region that mayexist as native genes or transgenes in a plant genome. The gene or afragment thereof is isolated and subjected to polynucleotide sequencingmethods that determines the order of the nucleotides that comprise thegene. Any of the components of the gene are potential targets for atrigger oligonucleotide and polynucleotides.

The trigger polynucleotide molecules are designed to modulate expressionby inducing regulation or suppression of an endogenous PPG oxidase genein a plant and are designed to have a nucleotide sequence essentiallyidentical or essentially complementary to the nucleotide sequence of anendogenous PPG oxidase gene of a plant or to the sequence of RNAtranscribed from an endogenous PPG oxidase gene of a plant, including atransgene in a plant that provides for a herbicide resistant PPG oxidaseenzyme, which can be coding sequence or non-coding sequence. Effectivemolecules that modulate expression are referred to as “a triggermolecule, or trigger polynucleotides”. By “essentially identical” or“essentially complementary” is meant that the trigger polynucleotides(or at least one strand of a double-stranded polynucleotide or portionthereof, or a portion of a single strand polynucleotide) are designed tohybridize to the endogenous gene noncoding sequence or to RNAtranscribed (known as messenger RNA or an RNA transcript) from theendogenous gene to effect regulation or suppression of expression of theendogenous gene. Trigger molecules are identified by “tiling” the genetargets with partially overlapping probes or non-overlapping probes ofantisense or sense polynucleotides that are essentially identical oressentially complementary to the nucleotide sequence of an endogenousgene. Multiple target sequences can be aligned and sequence regions withhomology in common, according to the methods, are identified aspotential trigger molecules for the multiple targets. Multiple triggermolecules of various lengths, for example 18-25 nucleotides, 26-50nucleotides, 51-100 nucleotides, 101-200 nucleotides, 201-300nucleotides or more can be pooled into a few treatments in order toinvestigate polynucleotide molecules that cover a portion of a genesequence (for example, a portion of a coding versus a portion of anoncoding region, or a 5′ versus a 3′ portion of a gene) or an entiregene sequence including coding and noncoding regions of a target gene.Polynucleotide molecules of the pooled trigger molecules can be dividedinto smaller pools or single molecules inorder to identify triggermolecules that provide the desired effect.

The target gene RNA and DNA polynucleotide molecules (Table 1, SEQ IDNO:1-71) are sequenced by any number of available methods and equipment.Some of the sequencing technologies are available commercially, such asthe sequencing-by-hybridization platform from Affymetrix Inc.(Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) andHelicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligationplatform from Applied Biosystems (Foster City, Calif), as describedbelow. In addition to the single molecule sequencing performed usingsequencing-by-synthesis of Helicos Biosciences, other single moleculesequencing technologies are encompassed by the method and include theSMRT™ . . . technology of Pacific Biosciences, the Ion Torrent™technology, and nanopore sequencing being developed for example, byOxford Nanopore Technologies. A PPG oxidase target gene comprising DNAor RNA can be isolated using primers or probes essentially complementaryor essentially homologous to SEQ ID NO:1-71 or a fragment thereof Apolymerase chain reaction (PCR) gene fragment can be produced usingprimers essentially complementary or essentially homologous to SEQ IDNO:1-71 or a fragment thereof that is useful to isolate a PPG oxidasegene from a plant genome. SEQ ID NO: 1-71 or fragments thereof can beused in various sequence capture technologies to isolate additionaltarget gene sequences, for example, including but not limited to RocheNimbleGen® (Madison, Wis.) and Streptavdin-coupled Dynabeads® (LifeTechnologies, Grand Island, N.Y.) and US20110015284, herein incorporatedby reference in its entirety.

Embodiments of functional single-stranded polynucleotides have sequencecomplementarity that need not be 100 percent, but is at least sufficientto permit hybridization to RNA transcribed from the target gene or DNAof the target gene to form a duplex to permit a gene silencingmechanism. Thus, in embodiments, a polynucleotide fragment is designedto be essentially identical to, or essentially complementary to, asequence of 18 or more contiguous nucleotides in either the target PPGoxidase gene sequence or messenger RNA transcribed from the target gene.By “essentially identical” is meant having 100 percent sequence identityor at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, or 99 percent sequence identity when compared to thesequence of 18 or more contiguous nucleotides in either the target geneor RNA transcribed from the target gene; by “essentially complementary”is meant having 100 percent sequence complementarity or at least about83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99percent sequence complementarity when compared to the sequence of 18 ormore contiguous nucleotides in either the target gene or RNA transcribedfrom the target gene. In some embodiments, polynucleotide molecules aredesigned to have 100 percent sequence identity with or complementarityto one allele or one family member of a given target gene (coding ornon-coding sequence of a gene); in other embodiments the polynucleotidemolecules are designed to have 100 percent sequence identity with orcomplementarity to multiple alleles or family members of a given targetgene. The trigger polynucleotide sequences in the sequence listing SEQID NO: 1-2221 or table 1, 2 or 3 maybe complementary or homologous to aportion of the PPG oxidase target gene sequence.

In certain embodiments, the polynucleotides used in the compositionsthat are essentially identical or essentially complementary to thetarget gene or transcript will comprise the predominant nucleic acid inthe composition. Thus in certain embodiments, the polynucleotides thatare essentially identical or essentially complementary to the targetgene or transcript will comprise at least about 50%, 75%, 95%, 98% or100% of the nucleic acids provided in the composition by either mass ormolar concentration. However, in certain embodiments, thepolynucleotides that are essentially identical or essentiallycomplementary to the target gene or transcript can comprise at leastabout 1% to about 50%, about 10% to about 50%, about 20% to about 50%,or about 30% to about 50% of the nucleic acids provided in thecomposition by either mass or molar concentration. Also provided arecompositions where the polynucleotides that are essentially identical oressentially complementary to the target gene or transcript can compriseat least about 1% to 100%, about 10% to 100%, about 20% to about 100%,about 30% to about 50%, or about 50% to a 100% of the nucleic acidsprovided in the composition by either mass or molar concentration.

“Identity” refers to the degree of similarity between two polynucleicacid or protein sequences. An alignment of the two sequences isperformed by a suitable computer program. A widely used and acceptedcomputer program for performing sequence alignments is CLUSTALW v1.6(Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number ofmatching bases or amino acids is divided by the total number of bases oramino acids, and multiplied by 100 to obtain a percent identity. Forexample, if two 580 base pair sequences had 145 matched bases, theywould be 25 percent identical. If the two compared sequences are ofdifferent lengths, the number of matches is divided by the shorter ofthe two lengths. For example, if there are 100 matched amino acidsbetween a 200 and a 400 amino acid protein, they are 50 percentidentical with respect to the shorter sequence. If the shorter sequenceis less than 150 bases or 50 amino acids in length, the number ofmatches are divided by 150 (for nucleic acid bases) or 50 (for aminoacids), and multiplied by 100 to obtain a percent identity.

Trigger molecules for specific gene family members can be identifiedfrom coding and/or non-coding sequences of gene families of a plant ormultiple plants, by aligning and selecting 200-300 polynucleotidefragments from the least homologous regions amongst the alignedsequences and evaluated using topically applied polynucleotides (assense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine theirrelative effectiveness in inducing the herbicidal phenotype. Theeffective segments are further subdivided into 50-60 polynucleotidefragments, prioritized by least homology, and reevaluated usingtopically applied polynucleotides. The effective 50-60 polynucleotidefragments are subdivided into 19-30 polynucleotide fragments,prioritized by least homology, and again evaluated for induction of theyield/quality phenotype. Once relative effectiveness is determined, thefragments are utilized singly, or again evaluated in combination withone or more other fragments to determine the trigger composition ormixture of trigger polynucleotides for providing the yield/qualityphenotype.

Trigger molecules for broad activity can be identified from codingand/or non-coding sequences of gene families of a plant or multipleplants, by aligning and selecting 200-300 polynucleotide fragments fromthe most homologous regions amongst the aligned sequences and evaluatedusing topically applied polynucleotides (as sense or anti-sense ssDNA orssRNA, dsRNA, or dsDNA) to determine their relative effectiveness ininducing the yield/quality phenotype. The effective segments aresubdivided into 50-60 polynucleotide fragments, prioritized by mosthomology, and reevaluated using topically applied polynucleotides. Theeffective 50-60 polynucleotide fragments are subdivided into 19-30polynucleotide fragments, prioritized by most homology, and againevaluated for induction of the yield/quality phenotype. Once relativeeffectiveness is determined, the fragments may be utilized singly, or incombination with one or more other fragments to determine the triggercomposition or mixture of trigger polynucleotides for providing theyield/quality phenotype.

Methods of making polynucleotides are well known in the art. Chemicalsynthesis, in vivo synthesis and in vitro synthesis methods andcompositions are known in the art and include various viral elements,microbial cells, modified polymerases, and modified nucleotides.Commercial preparation of oligonucleotides often provides twodeoxyribonucleotides on the 3′ end of the sense strand. Longpolynucleotide molecules can be synthesized from commercially availablekits, for example, kits from Applied Biosystems/Ambion (Austin, Tex.)have DNA ligated on the 5′ end in a microbial expression cassette thatincludes a bacterial T7 polymerase promoter that makes RNA strands thatcan be assembled into a dsRNA and kits provided by various manufacturersthat include T7 RiboMax Express (Promega, Madison, Wis.), AmpliScribeT7-Flash (Epicentre, Madison, Wis.), and TranscriptAid T7 High Yield(Fermentas, Glen Burnie, Md.). dsRNA molecules can be produced frommicrobial expression cassettes in bacterial cells (Ongvarrasopone et al.ScienceAsia 33:35-39; Yin, Appl. Microbiol. Biotechno184:323-333, 2009;Liu et al., BMC Biotechnology 10:85, 2010) that have regulated ordeficient RNase III enzyme activity or the use of various viral vectorsto produce sufficient quantities of dsRNA. PPG oxidase gene fragmentsare inserted into the microbial expression cassettes in a position inwhich the fragments are express to produce ssRNA or dsRNA useful in themethods described herein to regulate expression on a target PPG oxidasegene. Long polynucleotide molecules can also be assembled from multipleRNA or DNA fragments. In some embodiments design parameters such asReynolds score (Reynolds et al. Nature Biotechnology 22, 326-330(2004),Tuschl rules (Pei and Tuschl, Nature Methods 3(9): 670-676,2006), i-score (Nucleic Acids Res 35: e123, 2007), i-Score Designer tooland associated algorithms (Nucleic Acids Res 32: 936-948, 2004. BiochemBiophys Res Commun 316: 1050-1058, 2004, Nucleic Acids Res 32: 893-901,2004, Cell Cycle 3: 790-5, 2004, Nat Biotechnol 23: 995-1001, 2005,Nucleic Acids Res 35: e27, 2007, BMC Bioinformatics 7: 520, 2006,Nucleic Acids Res 35: e123, 2007, Nat Biotechnol 22: 326-330, 2004) areknown in the art and may be used in selecting polynucleotide sequenceseffective in gene silencing. In some embodiments the sequence of apolynucleotide is screened against the genomic DNA of the intended plantto minimize unintentional silencing of other genes.

The trigger polynucleotide and oligonucleotide molecule compositions areuseful in compositions, such as liquids that comprise thesepolynucleotide molecules, at low concentrations, alone or in combinationwith other components, for example one or more herbicide molecules,either in the same solution or in separately applied liquids that alsoprovide a transfer agent. While there is no upper limit on theconcentrations and dosages of polynucleotide molecules that can usefulin the methods, lower effective concentrations and dosages willgenerally be sought for efficiency. The concentrations can be adjustedin consideration of the volume of spray or treatment applied to plantleaves or other plant part surfaces, such as flower petals, stems,tubers, fruit, anthers, pollen, or seed. In one embodiment, a usefultreatment for herbaceous plants using 25-mer oligonucleotide moleculesis about 1 nanomole (nmol) of oligonucleotide molecules per plant, forexample, from about 0.05 to 1 nmol per plant. Other embodiments forherbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol ofpolynucleotides per plant. Very large plants, trees, or vines mayrequire correspondingly larger amounts of polynucleotides. When usinglong dsRNA molecules that can be processed into multipleoligonucleotides, lower concentrations can be used. To illustratecertain embodiments, the factor 1X, when applied to oligonucleotidemolecules is arbitrarily used to denote a treatment of 0.8 nmol ofpolynucleotide molecule per plant; 10×, 8 nmol of polynucleotidemolecule per plant; and 100×, 80 nmol of polynucleotide molecule perplant.

The polynucleotide compositions are useful in compositions, such asliquids that comprise polynucleotide molecules, alone or in combinationwith other components either in the same liquid or in separately appliedliquids that provide a transfer agent. As used herein, a transfer agentis an agent that, when combined with a polynucleotide in a compositionthat is topically applied to a target plant surface, enables thepolynucleotide to enter a plant cell. In certain embodiments, a transferagent is an agent that conditions the surface of plant tissue, e.g.,leaves, stems, roots, flowers, or fruits, to permeation by thepolynucleotide molecules into plant cells. The transfer ofpolynucleotides into plant cells can be facilitated by the prior orcontemporaneous application of a polynucleotide-transferring agent tothe plant tissue. In some embodiments the transferring agent is appliedsubsequent to the application of the polynucleotide composition. Thepolynucleotide transfer agent enables a pathway for polynucleotidesthrough cuticle wax barriers, stomata and/or cell wall or membranebarriers into plant cells. Suitable transfer agents to facilitatetransfer of the polynucleotide into a plant cell include agents thatincrease permeability of the exterior of the plant or that increasepermeability of plant cells to oligonucleotides or polynucleotides. Suchagents to facilitate transfer of the composition into a plant cellinclude a chemical agent, or a physical agent, or combinations thereof.Chemical agents for conditioning or transfer include (a) surfactants,(b) an organic solvent or an aqueous solution or aqueous mixtures oforganic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils,(g) enzymes, or combinations thereof. Embodiments of the method canoptionally include an incubation step, a neutralization step (e.g., toneutralize an acid, base, or oxidizing agent, or to inactivate anenzyme), a rinsing step, or combinations thereof. Embodiments of agentsor treatments for conditioning of a plant to permeation bypolynucleotides include emulsions, reverse emulsions, liposomes, andother micellar-like compositions. Embodiments of agents or treatmentsfor conditioning of a plant to permeation by polynucleotides includecounter-ions or other molecules that are known to associate with nucleicacid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions,lithium ions, polyamines such as spermine, spermidine, or putrescine,and other cations. Organic solvents useful in conditioning a plant topermeation by polynucleotides include DMSO, DMF, pyridine,N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane,polypropylene glycol, other solvents miscible with water or that willdissolve phosphonucleotides in non-aqueous systems (such as is used insynthetic reactions). Naturally derived or synthetic oils with orwithout surfactants or emulsifiers can be used, e.g., plant-sourcedoils, crop oils (such as those listed in the 9^(th) Compendium ofHerbicide Adjuvants, publicly available on the worldwide web (internet)at herbicide.adjuvants.com can be used, e.g., paraffinic oils, polyolfatty acid esters, or oils with short-chain molecules modified withamides or polyamines such as polyethyleneimine or N-pyrrolidine.Transfer agents include, but are not limited to, organosiliconepreparations.

Ligands can be tethered to a polynucleotide, for example a dsRNA, ssRNA,dsDNA or ssDNA. Ligands in general can include modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophiles, lipids (e.g., cholesterol, a bile acid, ora fatty acid (e.g., lithocholic-oleyl, lauroyl, docosnyl, stearoyl,palmitoyl, myristoyl oleoyl, linoleoyl), steroids (e.g., uvaol,hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics. Theligand may also be a recombinant or synthetic molecule, such as asynthetic polymer, e.g., polyethylene glycol (PEG), PEG-40K, PEG-20K andPEG-5K. Other examples of ligands include lipophilic molecules, e.g,cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, glycerol (e.g., esters and ethers thereof, e.g.,C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16,C.sub.17, C.sub.18, C.sub.19, or C.sub.20 alkyl; e.g., lauroyl,docosnyl, stearoyl, oleoyl, linoleoyl 1,3-bis-0(hexadecyl)glycerol,1,3-bis-0(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dodecanoyl, lithocholyl, 5.beta.-cholanyl,N,N-distearyl-lithocholamide, 1,2-di-O-stearoylglyceride,dimethoxytrityl, or phenoxazine) and PEG (e.g., PEG-5K, PEG-20K,PEG-40K). Preferred lipophilic moieties include lipid, cholesterols,oleyl, retinyl, or cholesteryl residues.

Conjugating a ligand to a dsRNA can enhance its cellular absorption,lipophilic compounds that have been conjugated to oligonucleotidesinclude 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, andmenthol. One example of a ligand for receptor-mediated endocytosis isfolic acid. Folic acid enters the cell by folate-receptor-radiatedendocytosis. dsRNA compounds bearing folic acid would be efficientlytransported into the cell via the folate-receptor-mediated endocytosis.Other ligands that have been conjugated to oligonucleotides includepolyethylene glycols, carbohydrate clusters, cross-linking agents,porphyrin conjugates, delivery peptides and lipids such as cholesterol.In certain instances, conjugation of a cationic ligand tooligonucleotides results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed, throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

A biologic delivery can be accomplished by a variety of methodsincluding, without limitation, (1) loading liposomes with a dsRNA acidmolecule provided herein and (2) complexing a dsRNA molecule with lipidsor liposomes to form nucleic acid-lipid or nucleic acid-liposomecomplexes. The liposome can be composed of cationic and neutral lipidscommonly used to transfect cells in vitro. Cationic lipids can complex(e.g., charge-associate) with negatively charged, nucleic acids to formliposomes. Examples of cationic liposomes include, without limitation,lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures forforming liposomes are well known in the art. Liposome compositions canbe formed, for example, from phosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoylphosphatidyl glycerol, dioleoyl phosphatidylethanolamine or liposomescomprising dihydrosphingomyelin (DHSM) Numerous lipophilic agents arecommercially available, including Lipofectin.®. (Invitrogen/LifeTechnologies, Carlsbad, Calif.) and Effectene™ (Qiagen, Valencia,Calif.), In addition, systemic delivery methods can be optimized usingcommercially available cationic lipids such as DDAB or DOTAP, each ofwhich can be mixed with a neutral lipid such as DOPE or cholesterol. Insome eases, liposomes such as those described by Templeton et al. NatureBiotechnology, 15:647-652 (1997) can be used. In other embodiments,polycations such as polyethyleneimine can be used to achieve delivery invivo and ex vivo (Boletta et al., J. Am Soc. Nephrol. 7:1728, 1996).Additional information regarding the use of liposomes to deliver nucleicacids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO96/40964 and Morrissey, D. et al., 2005, Nature Biotechnol.23(8):1002-7.

In certain embodiments, an organosilicone preparation that iscommercially available as Silwet® L-77 surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currentlyavailable from Momentive Performance Materials, Albany, N.Y. can be usedto prepare a polynucleotide composition. In certain embodiments where aSilwet L-77 organosilicone preparation is used as a pre-spray treatmentof plant leaves or other plant surfaces, freshly made concentrations inthe range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious inpreparing a leaf or other plant surface for transfer of polynucleotidemolecules into plant cells from a topical application on the surface. Incertain embodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01,0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065,0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.5 wt percent) is used or provided.

In certain embodiments, any of the commercially available organosiliconepreparations provided such as the following Breakthru S 321, Breakthru S200 Cat# 67674-67-3, Breakthru OE 441 Cat#68937-55-3, Breakthru S 278Cat #27306-78-1, Breakthru S 243, Breakthru S 233 Cat#134180-76-0,available from manufacturer Evonik Goldschmidt (Germany), Silwet® HS429, Silwet® HS 312, Silwet® HS 508, Silwet® HS 604 (MomentivePerformance Materials, Albany, N.Y.) can be used as transfer agents in apolynucleotide composition. In certain embodiments where anorganosilicone preparation is used as a pre-spray treatment of plantleaves or other surfaces, freshly made concentrations in the range ofabout 0.015 to about 2 percent by weight (wt percent) (e. g., about0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06,0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leafor other plant surface for transfer of polynucleotide molecules intoplant cells from a topical application on the surface. In certainembodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation in the range of about 0.015 to about 2percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025,0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wtpercent) is used or provided.

Organosilicone preparations used in the methods and compositionsprovided herein can comprise one or more effective organosiliconecompounds. As used herein, the phrase “effective organosiliconecompound” is used to describe any organosilicone compound that is foundin an organosilicone preparation that enables a polynucleotide to entera plant cell. In certain embodiments, an effective organosiliconecompound can enable a polynucleotide to enter a plant cell in a mannerpermitting a polynucleotide mediated suppression of a target geneexpression in the plant cell. In general, effective organosiliconecompounds include, but are not limited to, compounds that can comprise:i) a trisiloxane head group that is covalently linked to, ii) an alkyllinker including, but not limited to, an n-propyl linker, that iscovalently linked to, iii) a poly glycol chain, that is covalentlylinked to, iv) a terminal group. Trisiloxane head groups of sucheffective organosilicone compounds include, but are not limited to,heptamethyltrisiloxane. Alkyl linkers can include, but are not limitedto, an n-propyl linker Poly glycol chains include, but are not limitedto, polyethylene glycol or polypropylene glycol. Poly glycol chains cancomprise a mixture that provides an average chain length “n” of about“7.5”. In certain embodiments, the average chain length “n” can varyfrom about 5 to about 14. Terminal groups can include, but are notlimited to, alkyl groups such as a methyl group. Effectiveorganosilicone compounds are believed to include, but are not limitedto, trisiloxane ethoxylate surfactants or polyalkylene oxide modifiedheptamethyl trisiloxane.

In certain embodiments, an organosilicone preparation that comprises anorganosilicone compound comprising a trisiloxane head group is used inthe methods and compositions provided herein. In certain embodiments, anorganosilicone preparation that comprises an organosilicone compoundcomprising a heptamethyltrisiloxane head group is used in the methodsand compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments of themethods and compositions provided herein, a composition that comprises apolynucleotide molecule and one or more effective organosiliconecompound in the range of about 0.015 to about 2 percent by weight (wtpercent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used orprovided.

Compositions include but are not limited components that are one or morepolynucleotides essentially identical to, or essentially complementaryto a PPG oxidase gene sequence (promoter, intron, exon, 5′ untranslatedregion, 3′ untranslated region), a transfer agent that provides for thepolynucleotide to enter a plant cell, a herbicide that complements theaction of the polynucleotide, one or more additional herbicides thatfurther enhance the herbicide activity of the composition or provide anadditional mode of action different from the complementing herbicide,various salts and stabilizing agents that enhance the utility of thecomposition as an admixture of the components of the composition.

The methods include one or more applications of a polynucleotidecomposition and one or more applications of a permeability-enhancingagent for conditioning of a plant to permeation by polynucleotides. Whenthe agent for conditioning to permeation is an organosiliconecomposition or compound contained therein, embodiments of thepolynucleotide molecules are double-stranded RNA oligonucleotides,single-stranded RNA oligonucleotides, double-stranded RNApolynucleotides, single-stranded RNA polynucleotides, double-strandedDNA oligonucleotides, single-stranded DNA oligonucleotides,double-stranded DNA polynucleotides, single-stranded DNApolynucleotides, chemically modified RNA or DNA oligonucleotides orpolynucleotides or mixtures thereof.

Compositions and methods are useful for modulating the expression of anendogenous PPG oxidase gene (for example U.S. Pat. Nos. 7,838,263;6,084,155) or transgenic PPG oxidase gene (U.S. Pat. Nos. 7,842,856;7,485,777; US Patent Publ. 20070050863) in a plant cell. In variousembodiments, a PPG oxidase gene includes coding (protein-coding ortranslatable) sequence, non-coding (non-translatable) sequence, or bothcoding and non-coding sequence. Compositions can include polynucleotidesand oligonucleotides designed to target multiple genes, or multiplesegments of one or more genes. The target gene can include multipleconsecutive segments of a target gene, multiple non-consecutive segmentsof a target gene, multiple alleles of a target gene, or multiple targetgenes from one or more species.

A method is provided for modulating expression of a PPG oxidase gene ina plant including (a) conditioning of a plant to permeation bypolynucleotides and (b) treatment of the plant with the polynucleotidemolecules, wherein the polynucleotide molecules include at least onesegment of 18 or more contiguous nucleotides cloned from or otherwiseidentified from the target PPG oxidase gene in either anti-sense orsense orientation, whereby the polynucleotide molecules permeate theinterior of the plant and induce modulation of the target gene. Theconditioning and polynucleotide application can be performed separatelyor in a single step. When the conditioning and polynucleotideapplication are performed in separate steps, the conditioning canprecede or can follow the polynucleotide application within minutes,hours, or days. In some embodiments more than one conditioning step ormore than one polynucleotide molecule application can be performed onthe same plant. In embodiments of the method, the segment can be clonedor identified from (a) coding (protein-encoding), (b) non-coding(promoter and other gene related molecules), or (c) both coding andnon-coding parts of the target gene. Non-coding parts include DNA, suchas promoter regions or the RNA transcribed by the DNA that provide RNAregulatory molecules, including but not limited to: introns, 5′ or 3′untranslated regions, and microRNAs (miRNA), trans-acting siRNAs,natural anti-sense siRNAs, and other small RNAs with regulatory functionor RNAs having structural or enzymatic function including but notlimited to: ribozymes, ribosomal RNAs, t-RNAs, aptamers, andriboswitches.

All publications, patents and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The following examples are included to demonstrate examples of certainpreferred embodiments. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples ofpreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLES Example 1 Polynucleotides Related to the PPG Oxidase GeneSequences

The target PPG oxidase polynucleotide molecule naturally occurs in thegenome of Amaranthus albus, Amaranthus graecizans, Amaranthus hybridus,Amaranthus lividus, Amaranthus palmeri, Amaranthus rudis, Amaranthusspinosus, Amaranthus thunbergii, Amaranthus viridis, Ambrosia trifida,Chenopodium album, Commelina diffusa, Conyza canadensis, Digitariasanguinalis, Euphorbia heterophylla, Kochia scoparia, Lolium multiflorumand include molecules related to the expression of a polypeptideidentified as a PPG oxidase, that include regulatory molecules, cDNAscomprising coding and noncoding regions of a PPG oxidase gene andfragments thereof as shown in Table 1.

Polynucleotide molecules were extracted from these plant species bymethods standard in the field, for example, total RNA was extractedusing Trizol Reagent (Invitrogen Corp, Carlsbad, Calif. Cat. No.15596-018), following the manufacturer's protocol or modificationsthereof by those skilled in the art of polynucleotide extraction thatmay enhance recover or purity of the extracted RNA. Briefly, start with1 gram of ground plant tissue for extraction. Prealiquot 10 milliliters(mL) Trizol reagent to 15 mL conical tubes. Add ground powder to tubesand shake to homogenize. Incubate the homogenized samples for 5 minutes(min) at room temperature (RT) and then add 3 ml, of chloroform. Shakestubes vigorously by hand for 15-30 seconds(sec) and incubate at RT for 3min. Centrifuge the tubes at 7,000 revolutions per minute (rpm) for 10min at 4 degrees C. Transfer the aqueous phase to a new 1.5 mL tube andadd 1 volume of cold isopropanol. Incubate the samples for 20-30 min atRT and centrifuge at 10,000 rpm for 10 min at 4 degrees C. Wash pelletwith Sigma-grade 80 percent ethanol. Remove the supernatant and brieflyair-dry the pellet. Dissolve the RNA pellet in approximately 200microliters of DEPC treated water. Heat briefly at 65 degrees C. todissolve pellet and vortex or pipet to resuspend RNA pellet. Adjust RNAconcentraiton to 1-2 microgram/microliter.

DNA was extracted using EZNA SP Plant DNA Mini kit (Omega Biotek,Norcross Ga., Cat#D5511) and Lysing Matrix E tubes (Q-Biogen, Cat#6914),following the manufacturer's protocol or modifications thereof by thoseskilled in the art of polynucleotide extraction that may enhance recoveror purity of the extracted DNA. Briefly, aliquot ground tissue to aLysing Matrix E tube on dry ice, add 800 μl Buffer SP1 to each sample,homogenize in a bead beater for 35-45sec, incubate on ice for 45-60 sec,centrifuge at >14000 rpm for 1 min at RT, add 10 microliter RNase A tothe lysate, incubate at 65° C. for 10min, centrifuge for 1 min at RT,add 280 μl Buffer SP2 and vortex to mix, incubate the samples on ice for5 min, centrifuge at ≧10,000g for 10 min at RT, transfer the supernatantto a homogenizer column in a 2 ml collection tube, centrifuge at 10,000gfor 2 min at RT, transfer the cleared lysate into a 1.5 ml microfugetube, add 1.5 volumes Buffer SP3 to the cleared lysate, vorteximmediately to obtain a homogeneous mixture, transfer up to 650 μlsupernatant to the Hi-Bind column, centrifuge at 10,000g for 1 min,repeat, apply 100 μl 65° C. Elution Buffer to the column, centrifuge at10,000g for 5 min at RT.

Next-generation DNA sequencers, such as the 454-FLX (Roche, Branford,Conn.), the SOLiD (Applied Biosystems,), and the Genome Analyzer(HiSeq2000, Illumina, San Diego, Calif.) were used to providepolynucleotide sequence from the DNA and RNA extracted from the planttissues. Raw sequence data is assembled into contigs. The contigsequence is used to identify trigger molecules that can be applied tothe plant to enable regulation of the gene expression.

The target DNA sequence isolated from genomic (gDNA) and coding DNA(cDNA) from the various weedy plant species for the PPG oxidase gene andthe assembled contigs as set forth in SEQ ID NOs 1-71 and Table 1 (Seesupplemental attachment 40_(—)21(58640)Btable1.doxc)

Example 2 Polynucleotides Related to the Trigger Molecules

The gene sequences and fragments of Table 1 were divided into 200polynucleotide (200-mer) lengths with 25 polynucleotide overlappingregions and are shown in Table 2, SEQ ID NO:72-429. Thesepolynucleotides are tested to select the most efficacious triggerregions across the length of any target sequence. The triggerpolynucleotides are constructed as sense or anti-sense ssDNA or ssRNA,dsRNA, or dsDNA, or dsDNA/RNA hybrids and combined with anorganosilicone based transfer agent to provide a polynucleotidepreparation. The polynucleotides are combined into sets of two to threepolynucleotides per set, using 4-8 nmol of each polynucleotide. Eachpolynucleotide set is prepared with the transfer agent and applied to aplant or a field of plants in combination with a PPG oxidase inhibitorcontaining herbicide, or followed by a PPG oxidase inhibitor treatmentone to three days after the polynucleotide application, to determine theeffect on the plant's susceptibility to a PPG oxidase inhibitor. Theeffect is measured as stunting the growth and/or killing of the plantand is measured 8-14 days after treatment with the polynucleotide setand PPG oxidase inhibitor. The most efficacious sets are identified andthe individual polynucleotides are tested in the same methods as thesets are and the most efficacious single 200-mer identified. The 200-mersequence is divided into smaller sequences of 50-70-mer regions with10-15 polynucleotide overlapping regions and the polynucleotides testedindividually. The most efficacious 50-70-mer is further divided intosmaller sequences of 25-mer regions with a 12 to 13 polynucleotideoverlapping region and tested for efficacy in combination with PPGoxidase inhibitor treatment. By this method it is possible to identifyan oligonucleotide or several oligonucleotides that are the mostefficacious trigger molecule to effect plant sensitivity to a PPGoxidase inhibitor or modulation of a PPG oxidase gene expression. Themodulation of PPG oxidase gene expression is determined by the detectionof PPG oxidase siRNA moleclules specific to a PPG oxidase gene or by anobservation of a reduction in the amount of PPG oxidase RNA transcriptproduced relative to an untreated plant or by merely observing theanticipated phenotype of the application of the trigger with the PPGoxidase inhibitor containing herbicide. Detection of siRNA can beaccomplished, for example, using kits such as mirVana (Ambion, AustinTex.) and mirPremier (Sigma-Aldrich, St Louis, Mo.).

The target DNA sequence isolated from genomic (gDNA) and coding DNA(cDNA) from the various weedy plant species for the PPG oxidase gene andthe assembled contigs as set forth in SEQ ID NOs 1-71 were divided intopolynucleotide fragments as shown in Table 2 (See supplementalattachment 40_(—)21(58640)Btable2.docx) and as set forth in SEQ ID NOs72-1381.

The gene sequences and fragments of Table 1 were compared and 21-mers ofcontiguous polynucleotides were identified that had homology across thevarious PPG oxidase gene sequences. The purpose is to identify triggermolecules that are useful as herbicidal molecules or in combination witha PPG oxidase inhibitor herbicide across a broad range of weed species.The sequences shown in Table 3 represent the 21-mers that were presentin the PPG oxidase gene of at least eight of the weed species ofTable 1. It is contemplated that additional 21-mers can be selected fromthe sequences of Table 1 that are specific for a single weed species ora few weeds species within a genus or trigger molecules that are atleast 18 contiguous nucleotides, at least 19 contiguous nucleotides, atleast 20 contiguous nucleotides or at least 21 contiguous nucleotides inlength and at least 85 percent identical to a PPG oxidase gene sequenceselected from the group consisting of SEQ ID NO:1-71 or fragment thereof

By this method it is possible to identify an oligonucleotide or severaloligonucleotides that are the most efficacious trigger molecule toeffect plant sensitivity to PPG oxidase inhibitor or modulation of PPGoxidase gene expression. The modulation of PPG oxidase gene expressionis determined by the detection of PPG oxidase siRNA moleclules specificto PPG oxidase gene or by an observation of a reduction in the amount ofPPG oxidase RNA transcript produced relative to an untreated plant.Detection of siRNA can be accomplished, for example, using kits such asmirVana (Ambion, Austin Tex.) and mirPremier (Sigma-Aldrich, St Louis,Mo.).

The target DNA sequence isolated from genomic (gDNA) and coding DNA(cDNA) from the various weedy plant species for the PPG oxidase gene andthe assembled contigs as set forth in SEQ ID NOs 1-71 were divided intofragments as shown in Table 3 (See supplemental attachment 4021(58640)Btable3.docx) and as set forth in SEQ ID NOs 1382-2221.

Example 3 Methods Used Related to Treating Plants or Plant Parts with aTopical Mixture of the Trigger Molecules

Glyphosate-sensitive Palmer amaranth (A. palmeri R-22) plants were grownin the greenhouse (30/20 C day/night T; 14 hour photoperiod) in 4 inchsquare pots containing Sun Gro® Redi-Earth and 3.5 kg/cubic meterOsmocote® 14-14-14 fertilizer. Palmer amaranth plants at 5 to 10 cm inheight were pre-treated with a mixture of short (24-25mer) single-strandantisense oligo DNA polynucleotides (ssDNA,) targeting PPG oxidasecoding or noncoding regions at 16 nmol, formulated in 10 millimolarsodium phosphate buffer (pH 6.8) containing 2% ammonium sulfate and 0.5%Silwet L-77. Plants were treated manually by pipetting 10 μL ofpolynucleotide solution on four fully expanded mature leaves, for atotal of 40 microliters of solution per plant. Twenty-four andforty-eight hours later, the plants were treated with fomesafen (Reflex)at 22 g ai/ha and flumioxazin (Valor) at 4 g ai/ha, crop oil concentrate(COC) at 1% is added to the herbicide treatments. The formulationcontrol is the buffer and adjuvants without the DNA polynucleotides.Four replications of each treatment were conducted. Plant height isdetermined just before ssDNA treatment and at intervals upto fourteendays (dat) after herbicide treatments to determine effect of theoligonucleotide and herbicide treatments. The results shown in FIG. 1and FIG. 2 demonstrate that the oligonucleotide treatment enhanced theherbicidal activity of both fomesafen and flumioxazin.

A pool comprising eight antisense ssDNA oligonucleotides shown in Table4 was used to in the protocol as described to treat palmer amaranthplants and the individual oligonucleotides and various combinations ofoligonucleotides were tested for efficacy. Surprisingly, it wasnecessary to have a 3 oligonucleotide pool (oligo3, 5,and 7) of the 8oligonucleotides to reproduce the effect observed with the 8oligonucleotide pool and combinations of only 2 of the 3oligonucleotides was not effective to provide enhanced herbicidesensitivity. This result is illustrated in FIG. 3 for activity on Palmeramaranth, the same 3 oligonucleotides were also active on a relatedspecies, waterhemp (Amaranthus rudis).

TABLE 4 Antisense ssDNA PPG oxidase oligonucleotides OLIGO1SEQ ID NO: 2214 GTGATATTACCTCCAACACGAT OLIGO2 SEQ ID NO: 2215ATAGTAAGCACAGGATCGGAG OLIGO3 SEQ ID NO: 2216 CTTTCAATCCACTGTCAACCGOLIGO4 SEQ ID NO: 2217 ATCAAGCGTTCGAAGACCTCAT OLIGO5 SEQ ID NO: 2218CAGCAATGGCGGTAGGTAACA OLIGO6 SEQ ID NO: 2219 GCAATTGCCCGAATCCTTTTAOLIGO7 SEQ ID NO: 2220 TAGCTCAAtATCAAGGTCCTA OLIGO8 SEQ ID NO: 2221TCATAAGCACCCTCTATACAC

Example 4 A Method to Control Weeds in a Field

A method to control weeds in a field comprises the use of triggerpolynucleotides that can modulate the expression of a PPG oxidase genein one or more target weed plant species. In Table 3 (SEQ ID NO:1381-2221), an analysis of PPG oxidase gene sequences from seventeenplant species provided a collection of 21-mer polynucleotides that canbe used in compositions to affect the growth or develop or sensitivityto PPG oxidase inhibitor herbicide to control multiple weed species in afield. A composition containing 1 or 2 or 3 or 4 or more of thepolynucleotides of Table 3 would enable broad activity of thecomposition against the multiple weed species that occur in a fieldenvironment.

The method includes creating a composition that comprises componentsthat include at least one polynucleotide of Table 3 or any othereffective gene expression modulating polynucleotide essentiallyidentical or essentially complementary to SEQ ID NO:1-71 or fragmentthereof, a transfer agent that mobilizes the polynucleotide into a plantcell and a PPG oxidase inhibiting herbicide and optionally apolynucleotide that modulates the expression of an essential gene andoptionally a herbicide that has a different mode of action relative to aPPG oxidase inhibitor. The polynucleotide of the composition includes adsRNA, ssDNA or dsDNA or a combination thereof. A composition containinga polynucleotide can have a use rate of about 1 to 30 grams or more peracre depending on the size of the polynucleotide and the number ofpolynucleotides in the composition. The composition may include one ormore additional herbicides as needed to provide effective multi-speciesweed control. A field of crop plants in need of weed plant control istreated by spray application of the composition. The composition can beprovided as a tank mix, a sequential treatment of components (generallythe polynucleotide followed by the herbicide), a simultaneous treatmentor mixing of one or more of the components of the composition fromseparate containers. Treatment of the field can occur as often as neededto provide weed control and the components of the composition can beadjusted to target specific weed species or weed families.

Example 5 Herbicidal Compositions Comprising Pesticidal Agents

A method of controlling weeds and plant pest and pathogens in a field ofPPG oxidase inhibitor tolerant crop plants is provided, wherein themethod comprises applying a composition comprising a PPG oxidase triggeroligonucleotide, a PPG oxidase inhibitor composition and an admixture ofa pest control agent. For example, the admixture comprises insecticides,fungicides, nematocides, bactericides, acaricides, growth regulators,chemosterilants, semiochemicals, repellents, attractants, pheromones,feeding stimulants or other biologically active compounds or biologicalagents, such as, microorganisms.

For example, the admixture comprises a fungicide compound for use on aPPG oxidase inhibitor tolerant crop plant to prevent or control plantdisease caused by a plant fungal pathogen, The fungicide compound of theadmixture may be a systemic or contact fungicide or mixtures of each.More particularly the fungicide compound includes, but is not limited tomembers of the chemical groups strobilurins, triazoles, chloronitriles,carboxamides and mixtures thereof. The composition may additional havean admixture comprises an insecticidal compound or agent.

The PPG oxidase trigger oligonucleotides and PPG oxidase inhibitor (forexample, fomesafen) tank mixes with fungicides, insecticides or both aretested for use in soybean and corn for control of foliar diseases andpests. Testing is conducted to develop a method for use of mixtures ofthe trigger oligonucleotides and fomesafen formulation and variouscommercially available fungicides for weed control and pest control. Thefield plots are planted with soybeans or corn. All plots receive a postplant application of the PPG oxidase trigger+fomesafen about 3 weeksafter planting. The mixtures of trigger+fomesafen ortrigger+fomesafen+fungicide+insecticides are used to treat the plots atthe R1 stage of soybean development (first flowering) or tassel stage ofcorn. Data is taken for percent weed control at 7 and 21 days after R1treatment, soybean safety (% necrosis, chlorosis, growth rate): 5 daysafter treatment, disease rating, pest ratings and yield (bushels/Acre).These mixtures and treatments are designed to provide simultaneous weedand pest control, such as fungal pest control, for example, leaf rustdisease; and insect pest control, for example, aphids, armyworms,loopers, beetles, stinkbugs, and leaf hoppers.

Agricultural chemicals are provided in containers suitable for safestorage, transportation and distribution, stability of the chemicalcompositions, mixing with solvents and instructions for use. A containerof a mixture of a trigger oligonucleotide+a herbicice+fungicidecompound, or a mixture of a trigger oligonucleotide+herbicide compoundand an insecticide compound, or a trigger oligonucleotide+a herbicidecompound and a fungicide compound and an insecticide compound (forexample, lambda-cyhalothrin, Warriert). The container may furtherprovide instructions on the effective use of the mixture. Containers ofthe present invention can be of any material that is suitable for thestorage of the chemical mixture. Containers of the present invention canbe of any material that is suitable for the shipment of the chemicalmixture. The material can be of cardboard, plastic, metal, or acomposite of these materials. The container can have a volume of 0.5liter, 1 liter, 2 liter, 3-5 liter, 5-10 liter, 10-20 liter, 20-50 literor more depending upon the need. A tank mix of a triggeroligonucleotide+herbicide compound and a fungicide compound is provided,methods of application to the crop to achieve an effective dose of eachcompound are known to those skilled in the art and can be refined andfurther developed depending on the crop, weather conditions, andapplication equipment used.

Insecticides, fungicides, nematocides, bactericides, acaricides, growthregulators, chemosterilants, semiochemicals, repellents, attractants,pheromones, feeding stimulants or other biologically active compoundscan be added to the trigger oligonucleotide to form a multi-componentpesticide giving an even broader spectrum of agricultural protection.Examples of such agricultural protectants with which compounds of thisinvention can be formulated are: insecticides such as abamectin,acephate, azinphos-methyl, bifenthrin, buprofezin, carbofuran,chlorfenapyr, chlorpyrifos, chlorpyrifos-methyl, cyfluthrin,beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, deltamethrin,diafenthiuron, diazinon, diflubenzuron, dimethoate, esfenvalerate,fenoxycarb, fenpropathrin, fenvalerate, fipronil, flucythrinate,tau-fluvalinate, fonophos, imidacloprid, isofenphos, malathion,metaldehyde, methamidophos, methidathion, methomyl, methoprene,methoxychlor, methyl7-chloro-2,5-dihydro-2-[[N-(methoxycarbonyl)-N-[4-(trifluoromethoxy)phenyl]amino]carbonyl]indeno[1,2-e][1,3,4]oxadiazine-4a(3H)-carboxylate(DPX-JW062), monocrotophos, oxamyl, parathion, parathion-methyl,permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb,profenofos, rotenone, sulprofos, tebufenozide, tefluthrin, terbufos,tetrachlorvinphos, thiodicarb, tralomethrin, trichlorfon andtriflumuron; most preferably a PPG oxidase inhibitor compound isformulated with a fungicide compound or combinations of fungicides, suchas azoxystrobin, benomyl, blasticidin-S, Bordeaux mixture (tribasiccopper sulfate), bromuconazole, captafol, captan, carbendazim,chloroneb, chlorothalonil, copper oxychloride, copper salts, cymoxanil,cyproconazole, cyprodinil (CGA 219417), diclomezine, dicloran,difenoconazole, dimethomorph, diniconazole, diniconazole-M, dodine,edifenphos, epoxiconazole (BAS 480F), famoxadone, fenarimol,fenbuconazole, fenpiclonil, fenpropidin, fenpropimorph, fluazinam,fluquinconazole, flusilazole, flutolanil, flutriafol, folpet,fosetyl-aluminum, furalaxyl, hexaconazole, ipconazole, iprobenfos,iprodione, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb,maneb, mepronil, metalaxyl, metconazole, S-methyl7-benzothiazolecarbothioate (CGA 245704), myclobutanil, neo-asozin(ferric methanearsonate), oxadixyl, penconazole, pencycuron,probenazole, prochloraz, propiconazole, pyrifenox, pyroquilon,quinoxyfen, spiroxamine (KWG4168), sulfur, tebuconazole, tetraconazole,thiabendazole, thiophanate-methyl, thiram, triadimefon, triadimenol,tricyclazole, trifloxystrobin, triticonazole, validamycin andvinclozolin; combinations of fungicides are common for example,cyproconazole and azoxystrobin, difenoconazole, and metalaxyl-M,fludioxonil and metalaxyl-M, mancozeb and metalaxyl-M, copper hydroxideand metalaxyl-M, cyprodinil and fludioxonil, cyproconazole andpropiconazole; commercially available fungicide formulations for controlof Asian soybean rust disease include, but are not limited to Quadris®(Syngenta Corp), Bravo® (Syngenta Corp), Echo 720® (Sipcam Agro Inc),Headline® 2.09EC (BASF Corp), Tilt® 3.6EC (Syngenta Corp), PropiMax™3.6EC (Dow AgroSciences), Bumper® 41.8EC (MakhteshimAgan), Folicur® 3.6F(Bayer CropScience), Laredo® 25EC (Dow AgroSciences), Laredo™ 25EW (DowAgroSciences), Stratego® 2.08F (Bayer Corp), Domark™ 125SL (Sipcam AgroUSA), and Pristine®38% WDG (BASF Corp) these can be combined with PPGoxidase inhibitor compositions as described in the present invention toprovide enhanced protection from fungal disease; nematocides such asaldoxycarb and fenamiphos; bactericides such as streptomycin; acaricidessuch as amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol,dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin,fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; andbiological agents such as Bacillus thuringiensis, Bacillus thuringiensisdelta endotoxin, baculovirus, and entomopathogenic bacteria, virus andfungi.

Lengthy table referenced here US20130254941A1-20130926-T00001 Pleaserefer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130254941A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

We claim:
 1. A method of plant control comprising: treating a plant witha composition comprising a polynucleotide and a transfer agent, whereinsaid polynucleotide is essentially identical or essentiallycomplementary to a PPG oxidase gene sequence or fragment thereof, or toan RNA transcript of said PPG oxidase gene sequence or fragment thereof,wherein said PPG oxidase gene sequence is selected from the groupconsisting of SEQ ID NO:1-71 or a polynucleotide fragment thereof,whereby said plant growth or development or reproductive ability isregulated, suppressed or delayed or said plant is more sensitive to aPPG oxidase inhibitor herbicide as a result of said polynucleotidecontaining composition relative to a plant not treated with saidcomposition.
 2. The method as claimed in claim 1, wherein said transferagent is an organosilicone surfactant composition or compound containedtherein.
 3. The method as claimed in claim 1, wherein saidpolynucleotide fragment is 18 contiguous, 19 contiguous nucleotides, 20contiguous nucleotides or at least 21 contiguous nucleotides in lengthand at least 85 percent identical to a PPG oxidase gene sequenceselected from the group consisting of SEQ ID NO:1-71.
 4. The method asclaimed in claim 3, wherein said polynucleotide fragment is selectedfrom the group consisting of sense or anti-sense ssDNA or ssRNA, dsRNA,or dsDNA, or dsDNA/RNA hybrids.
 5. The method as claimed in claim 1,wherein said plant is selected from the group consisting of Amaranthusalbus, Amaranthus graecizans, Amaranthus hybridus, Amaranthus lividus,Amaranthus palmeri, Amaranthus rudis, Amaranthus spinosus, Amaranthusthunbergii, Amaranthus viridis, Ambrosia trifida, Chenopodium album,Commelina diffusa, Conyza canadensis, Digitaria sanguinalis, Euphorbiaheterophylla, Kochia scoparia, and Lolium multiflorum.
 6. The method asclaimed in claim 1, wherein said composition further comprises said PPGoxidase inhibitor herbicide and external application to a plant withsaid composition.
 7. The method as claimed in claim 6, wherein saidcomposition further comprises one or more herbicides different from saidPPG oxidase inhibitor herbicide.
 8. The method as claimed in claim 3,wherein said composition comprises any combination of two or more ofsaid polynucleotide fragments and external application to a plant withsaid composition.
 9. A composition comprising a polynucleotide and atransfer agent, wherein said polynucleotide is essentially identical oressentially complementary to a PPG oxidase gene sequence, or to an RNAtranscript of said PPG oxidase gene sequence, wherein said PPG oxidasegene sequence is selected from the group consisting of SEQ ID NO:1-71 ora polynucleotide fragment thereof and whereby a plant treated with saidcomposition has its growth or development or reproductive abilityregulated, suppressed or delayed or said plant is more sensitive to aPPG oxidase inhibitor herbicide or mitosis inhibitor herbicide as aresult of said polynucleotide containing composition relative to a plantnot treated with said composition.
 10. The composition of claim 9,wherein said transfer agent is an organosilicone composition.
 11. Thecomposition of claim 9, wherein said polynucleotide fragment is 18contiguous, 19 contiguous nucleotides, 20 contiguous nucleotides or atleast 21 contiguous nucleotides in length and at least 85 percentidentical to a PPG oxidase gene sequence selected from the groupconsisting of SEQ ID NO:1-71.
 12. The composition of claim 9, whereinsaid polynucleotide is selected from the group consisting of SEQ IDNO:72-1381.
 13. The composition of claim 9, wherein said polynucleotideis selected from the group consisting of SEQ ID NO: 1382-2221.
 14. Thecomposition of claim 9, further comprising a PPG oxidase inhibitorherbicide.
 15. The composition of claim 14, wherein said PPG oxidaseinhibitor molecule is selected from the group consisting ofacifluorfen-Na, bifenox, chlomethoxyfen, chlornitrofen,ethoxyfen-ethtyl, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen,oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin,flumiclorac-pentyl, fluthiacet-methyl, thidiazimin, oxadiazon,oxadiargyl, pyraclonil, flufenpyr-ethyl, azafenidin,carfentrazone-ethyl, Saflufenacil, sulfentrazone, pentoxazone.benzfendizone, butafenacil, pyrazogyl, and profluazol.
 16. Thecomposition of claim 14, further comprising a co-herbicide.
 17. A methodof reducing expression of a PPG oxidase gene in a plant comprising:external application to a plant of a composition comprising apolynucleotide and a transfer agent, wherein said polynucleotide isessentially identical or essentially complementary to a PPG oxidase genesequence or fragment thereof, or to the RNA transcript of said PPGoxidase gene sequence or fragment thereof, wherein said PPG oxidase genesequence is selected from the group consisting of SEQ ID NO:1-71 or apolynucleotide fragment thereof, whereby said expression of said PPGoxidase gene is reduced relative to a plant in which the composition wasnot applied.
 18. The method as claimed in claim 17, wherein saidtransfer agent is an organosilicone compound.
 19. The method as claimedin claim 17, wherein said polynucleotide fragment is 19 contiguousnucleotides, 20 contiguous nucleotides or at least 21 contiguousnucleotides in length and at least 85 percent identical to a PPG oxidasegene sequence selected from the group consisting of SEQ ID NO:1-71. 20.The method as claimed in 17, wherein said polynucleotide molecule isselected from the group consisting of sense or anti-sense ssDNA orssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.
 21. A microbial expressioncassette comprising a polynucleotide fragment of 18 contiguous, 19contiguous nucleotides, 20 contiguous nucleotides or at least 21contiguous nucleotides in length and at least 85 percent identical to aPPG oxidase gene sequence selected from the group consisting of SEQ IDNO:1-71.
 22. A method of making a polynucleotide comprising a)transforming the microbial expression cassette of claim 21 into amicrobe; b) growing said microbe; c) harvesting a polynucleotide fromsaid microbe, wherein said polynucleotide is at least 18 contiguous, atleast 19 contiguous nucleotides, at least 20 contiguous nucleotides orat least 21 contiguous nucleotides in length and at least 85 percentidentical to a PPG oxidase gene sequence selected from the groupconsisting of SEQ ID NO:1-71.
 23. A method of identifyingpolynucleotides useful in modulating PPG oxidase gene expression whenexternally treating a plant comprising: a) providing a plurality ofpolynucleotides that comprise a region essentially identical oressentially complementary to a polynucleotide fragment of 18 contiguous,19 contiguous nucleotides, 20 contiguous nucleotides or at least 21contiguous nucleotides in length and at least 85 percent identical to aPPG oxidase gene sequence selected from the group consisting of SEQ IDNO:1-71; b) externally treating said plant with one or more of saidpolynucleotides and a transfer agent; c) analyzing said plant or extractfor modulation of PPG oxidase gene expression, and whereby a planttreated with said composition has its growth or development orreproductive ability regulated, suppressed or delayed or said plant ismore sensitive to a PPG oxidase inhibitor herbicide or mitosis inhibitorherbicide as a result of said polynucleotide containing compositionrelative to a plant not treated with said composition.
 24. The method asclaimed in 23, wherein said plant is selected from the group consistingof Amaranthus albus, Amaranthus graecizans, Amaranthus hybridus,Amaranthus lividus, Amaranthus palmeri, Amaranthus rudis, Amaranthusspinosus, Amaranthus thunbergii, Amaranthus viridis, Ambrosia trifida,Chenopodium album, Commelina diffusa, Conyza canadensis, Digitariasanguinalis, Euphorbia heterophylla, Kochia scoparia, and Loliummultiflorum.
 25. The method as claimed in 23, wherein said PPG oxidasegene expression is reduced relative to a plant not treated with saidpolynucleotide fragment and a transfer agent.
 26. The method as claimedin 23, wherein said transfer agent is an organosilicone compound.
 27. Anagricultural chemical composition comprising an admixture of apolynucleotide and a PPG oxidase inhibitor herbicide and a co-herbicide,wherein said polynucleotide is essentially identical or essentiallycomplementary to a portion of a PPG oxidase gene sequence, or to aportion of an RNA transcript of said PPG oxidase gene sequence, whereinsaid PPG oxidase gene sequence is selected from the group consisting ofSEQ ID NO:1-71 or a polynucleotide fragment thereof, and whereby a planttreated with said composition has its growth or development orreproductive ability regulated, suppressed or delayed or said plant ismore sensitive to a PPG oxidase inhibitor herbicide or mitosis inhibitorherbicide as a result of said polynucleotide containing compositionrelative to a plant not treated with said composition.
 28. Theagricultural chemical composition of claim 27, wherein said co-herbicideis selected from the group consisting of amide herbicides, arsenicalherbicides, benzothiazole herbicides, benzoylcyclohexanedioneherbicides, benzofuranyl alkylsulfonate herbicides, cyclohexene oximeherbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides,dinitroaniline herbicides, dinitrophenol herbicides, dithiocarbamateherbicides, glycine herbicides, halogenated aliphatic herbicides,imidazolinone herbicides, inorganic herbicides, nitrile herbicides,organophosphorus herbicides, oxadiazolone herbicides, oxazoleherbicides, phenoxy herbicides, phenylenediamine herbicides, pyridazineherbicides, pyridazinone herbicides, pyridine herbicides,pyrimidinediamine herbicides, pyrimidinyloxybenzylamine herbicides,quaternary ammonium herbicides, thiocarbamate herbicides, thiocarbonateherbicides, thiourea herbicides, triazine herbicides, triazinoneherbicides, triazolone herbicides, triazolopyrimidine herbicides, uracilherbicides, and urea herbicides.
 29. An agricultural chemicalcomposition comprising an admixture of a polynucleotide and a PPGoxidase inhibitor herbicide and a pesticide, wherein said polynucleotideis essentially identical or essentially complementary to a portion of aPPG oxidase gene sequence, or to a portion of an RNA transcript of saidPPG oxidase gene sequence, wherein said PPG oxidase gene sequence isselected from the group consisting of SEQ ID NO:1-71 or a polynucleotidefragment thereof, whereby a field of crop plants in need of weed andpest control are treated with said composition, and whereby a planttreated with said composition has its growth or development orreproductive ability regulated, suppressed or delayed or said plant ismore sensitive to a PPG oxidase inhibitor herbicide or mitosis inhibitorherbicide as a result of said polynucleotide containing compositionrelative to a plant not treated with said composition.
 30. Theagricultural chemical composition of claim 29, wherein said pesticide isselected from the group consisting of insecticides, fungicides,nematocides, bactericides, acaricides, growth regulators,chemosterilants, semiochemicals, repellents, attractants, pheromones,feeding stimulants, and biopesticides.
 31. A herbicide mixturecomprising a PPG oxidase inhibitor and a polynucleotide mixturecomprising SEQ ID NO: 2216, 2218 and 2220, and whereby a plant treatedwith said composition has its growth or development or reproductiveability regulated, suppressed or delayed or said plant is more sensitiveto a PPG oxidase inhibitor herbicide or mitosis inhibitor herbicide as aresult of said polynucleotide containing composition relative to a plantnot treated with said composition.